Anti-cd40 binding molecules having engineered fc domains and therapeutic uses thereof

ABSTRACT

The disclosure provides CD40-binding molecules comprising engineered human IgG domains, for example, human IgG1, IgG2, and IgG4 variants having mutations in the hinge domain, which exhibit altered binding activity to one or more Fcγ receptors. Also described herein are methods for selectively activating or inhibiting immune responses in a subject using the CD40-binding molecules.

RELATED APPLICATION

This application is a Continuation of International Patent ApplicationNo. PCT/IB2019/001135, filed September 2019, which claims the benefit ofInternational Patent Application No. PCT/CN2018/0108285, filed on Sep.28, 2018, each of which is incorporated by reference herein in itsentirety.

BACKGROUND OF THE INVENTION

Cluster of differentiation 40 (CD40) is an antigen-presenting cell (APC)costimulatory protein required for APC activation. CD40 is a member ofthe tumor necrosis factor (TNF)-receptor superfamily and is essentialfor various immune and inflammatory responses, including Tcell-dependent immunoglobulin class switching, memory B celldevelopment, and germinal center formation. Additionally, CD40 is foundon the surface of tumor cells, such as B-lymphomas and about 70% of allsolid tumors. Its activation has been shown to reverse tolerance totumor-specific antigens, leading to antigen-specific antitumor immunity.

Fc receptors (FcR) are a family of immune cell surface proteins capableof binding to the Fc portion of antibodies. There are several differenttypes of Fc receptors, including Fcγ receptors, Fcα receptors, Fcεreceptors, and neonatal Fc receptors (FcRn), which have differentbinding activities to IgG, IgA, IgE, and IgG antibodies, respectively.The Fcγ receptor subfamily includes FcγRI (CD64), FcγRIIA (CD32a),FcγRIIB (CD32b), FcγRIIB (CD32c), FcγRIIIA (CD16a), and FcγRIIIB(CD16b). FcγRI has high binding affinity to IgG1 and IgG3 antibodies,while the other FcγRs have low binding affinity to IgG antibodies.

Different types of Fc receptors play different roles in the immunesystem. For example, FcγRIII receptors, expressed on NK cells andmacrophages, bind to antibodies that are attached to infected cells orinvading pathogens and trigger antibody-mediated phagocytosis (ADCP) orantibody-dependent cell-mediated cytotoxicity (ADCC) of the immunecells, thereby leading to elimination of the infected cells or invadingpathogens. On the other hand, FcγRIIB receptors, expressed on B cellsand dendritic cells, can down regulate the activity of the immune cellswhen binding to IgG antibodies.

Therapies involving activated immune cells are promising approaches foreliminating diseased cells such as cancer cells. However, suchtherapeutic approaches often raise safety concerns. For example, overlyactivated immune cells would lead to undesired cytotoxicity, causingtissue damage. It is therefore of great interest to develop new immunetherapies that are effective and safe.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the design ofCD40-binding molecules (e.g., CD40 agonists or CD40 antagonists)comprising an engineered (variant) Fc region (e.g., an engineered Fcregion of IgG1, IgG2, or IgG4 molecules) that exhibit desired hingeflexibility and preferred Fc receptor binding activity and/orselectivity, e.g., enhanced binding affinity and/or selected to FcγRIIBor substantially reduced binding affinity to one or more FcγR receptors.Such CD40-binding molecules can be used for modulating immune responses(e.g., selectively enhancing or selectively inhibiting an immuneresponse) in subject in need of the treatment.

Accordingly, one aspect of the present disclosure provides aCD40-binding molecule (e.g., a CD40 agonist or a CD40 antagonist)comprising a CD40 binding moiety and an antibody heavy chain constantregion comprising an engineered Fc region, which comprises at least onemutation at any of positions 220-331, for example, any of positions228-329, as compared to the wild-type Fc region counterpart. Thenumbering system is according to the EU index.

In some embodiments, the CD40 binding moiety is the extracellular domainof a CD40L, which may comprise the amino acid sequence of SEQ ID NO:140.A CD40-binding molecule comprising such a CD40-binding moiety may be aCD40 agonist.

In other embodiments, the CD40 binding moiety can be an anti-CD40antibody fragment. In one embodiment, the anti-CD40 antibody fragmentcomprises a heavy chain that comprises a heavy chain variable region,which is linked to any of the engineered Fc regions described herein,and a light chain that comprises a light chain variable region, and alight chain constant region.

In some instances, the variant Fc region may have an enhanced bindingaffinity and/or selectivity to FcγRIIB relative to the wild-typecounterpart. In other instances, the variant Fc region may havesubstantially reduced binding affinity to one or more FcγR receptors. Insome examples, the variant Fc region has low or no binding activity toall FcγR receptors.

In some embodiments, the CD40-binding molecule may comprise a variant Fcregion of an IgG1 molecule (e.g., a human IgG1 molecule), which maycomprise a mutation comprising one or more of the following: (a) anamino acid substitution or deletion within positions 233-238 (e.g.,234-238); (b) a substitution at position 265; (c) a substitution atposition 267; (d) a substitution at position 297; (e) a substitution atposition 328, a substitution at position 329; or a combination thereof.Alternatively or in addition, the Fc variant of an IgG1 molecule maycomprise one or more mutations at positions 220, 226, 229, 238, 273,327, 330, and/or 331. In one example, the mutation comprisessubstitutions at any of positions 233-236, a deletion at one or more ofpositions 236-238, or a combination thereof. For example, thesubstitutions at positions 233-235 may comprise E233P, L234V, L234F,L234A, L235A, and/or L235E. In one example, the mutation comprises adeletion at one or more of positions 236-238. In another example, thesubstitution at position 238 can be P238S, the substation at position265 can be D265A, or a combination thereof. In yet another example, thesubstitution is at position 267 and is S267E. In a further example, thesubstitution is at position 329 and is P329G. In an additional example,the mutation may comprise a substitution at position 265, a substitutionat position 297, or a combination thereof. For example, the substitutionat position 265 may be D265A and the substitution at position 297 may beN297A. Further, the one or more substitutions at positions 220, 226,229, 327, 330, and 331 can be C220S, C226S, C229S, A327G, A330S, andP331S. Exemplary variant Fc regions derived from human IgG1 may be oneof G1m1, G1m2, G1m17, G1m27, G1mAA, G1mAAG, G1N297A, G1m240, and G1m40.

In other embodiments, the CD40-binding molecule may comprise an Fcregion of an IgG2 molecule (e.g., a human IgG2 molecule), which maycomprise a mutation comprising one or more of the following: (a) adeletion of one or more of positions 237 and 238; (b) a substitution atposition 265; (b) a substitution at position 267; (c) a substitution atposition 297; (d) a substitution at position 328; or a combinationthereof. In some examples, the deletion is at position 237 or at bothpositions 237 and 238. Alternatively or in addition, the Fc variant ofan IgG2 molecule may comprise at least one mutation comprising asubstitution at one or more of positions 233-235, 237, 238, 268, 273,330, and 331. For example, the substitution at one or more of positions233-235, 237, 238, 268, 273, 330, and 331 can be P233E, V234A, V234L,A235L, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S. Inone example, the substitution is at position 267 and is S267E. Inanother example, the substitution is at position 328 and is L328F. In anadditional example, the mutation may comprise a substitution at position265, a substitution as position 297, or a combination thereof. Forexample, the substitution at position 265 may be D265A and thesubstitution at position 297 may be N297A. In some particular examples,the variant Fc region derived from IgG2 can be one of G2 m1, G2m17,G2m18, G2m19, G2m20, G2m27, G2m28, G2m29, G2m2040, G2m43, G2G4, G2mAA,and G2m40.

In yet other embodiments, the CD40-binding molecule may comprise an Fcregion of an IgG4 molecule (e.g., a human IgG4 molecule), which maycomprise (a) an amino acid residue substitution at position 228; (b) asubstitution or deletion at any one positions 235-238; (c) asubstitution at position 265; (d) a substitution at position 267; (e) asubstitution at position 297; (e) a substitution at position 328; or acombination thereof. In one example, the substitution at position 228 isS228P. In another example, the deletion is at one or more of positions236, 237, and 238 (e.g., at both positions 236 and 237). Alternativelyor in addition, the Fc variant of an IgG4 molecule may comprise asubstitution at one or more of positions 233-235, 237, and 273. Forexample, the substitution at one or more positions 233-235, 237, and 273may be E233P, F234V, F234A, L235S, L235E, L235A, G237A, and V273E. Inanother example, the substitution at position 267 is S267E. In a furtherexample, the substitution at position 328 is L328F. In an additionalexample, the mutation may comprise a substitution at position 265, asubstitution at position 297, or a combination thereof. For example, thesubstitution at position 265 may be D265A and the substitution atposition 297 may be N297A. In some particular examples, the variant Fcregion is one of G4 m1, G4m2, G4m20, G4m28, G4m30, G4m41, G4m42, G4m46,G4mPE, G4mAA, and G4m40.

Any of the variant Fc regions described herein may exhibit an enhancedbinding activity and/or an enhanced selectivity to FcγRIIB as comparedwith the wild-type Fc region. Alternatively, the variant Fc regionsdescribed herein may have low or no binding activity to any of the FcγRreceptors. In some instances, the variant Fc region may exhibit adecreased binding affinity to FcγRIIB. Alternatively or in addition, thevariant Fc region binds FcRn.

The anti-CD40 antibody fragment in any of the CD40-binding moleculesdescribed herein may be of a human antibody or a humanized antibody. Insome embodiments, the anti-CD40 antibody is an agonist antibody. In someexamples, the anti-CD40 antibody may comprise the same heavy chaincomplementary determining regions (HC CDRs) as those in SEQ ID NO: 128or as those in any one of 19G6D6, 36G7B8, 13F1A7, 9F12D9, and 17C5C2,and/or the same light chain complementary determining regions (LC CDRs)as those in SEQ ID NO: 129 or those in any one of 19G6D6, 36G7B8,13F1A7, 9F12D9, and 17C5C2. In one example, the antibody fragment maycomprise a heavy chain variable region of SEQ ID NO: 128 and/or a lightchain variable region of SEQ ID NO: 129. In other examples, the antibodyfragment may comprise the same heavy chain variable region and/or thesame light chain variable region as one of 19G6D6, 36G7B8, 13F1A7,9F12D9, and 17C5C2.

Additionally, provided herein is a pharmaceutical composition,comprising any of the CD40-binding molecules described herein and apharmaceutically acceptable carrier. Such a pharmaceutical compositionmay be used to selectively modulate (e.g., selectively activate orselectively inhibit) an immune response in a subject.

In yet another aspect, the present disclosure provides a method forselectively modulating (e.g., selectively activating or selectivelyinhibiting) an immune response in a subject, the method comprisingadministering to a subject in need thereof an effective amount of aCD40-binding molecule as described herein.

In any of the methods described herein, the subject can be a humanpatient having or suspected of having a cancer and the CD40-bindingmolecule may be a CD40 agonist. Exemplary cancers include lung cancer,stomach cancer, liver cancer, breast cancer, skin cancer, pancreaticcancer, brain cancer, prostate cancer, bladder cancer, colorectalcancer, sarcoma, bone cancer, lymphoma and a hematological cancer.

Alternatively, the subject can be a human patient having or suspected ofhaving an immune-related disorder and the CD40-binding molecule may be aCD40 antagonist. Exemplary immune disorders include autoimmune diseases,immune-deficiencies, or allergies. In some embodiments, the targetdisease for treatment is an autoimmune disease.

In yet another aspect, the disclosure provides an isolated anti-CD40antibody, which binds to the same epitope of CD40 as a referenceantibody selected from the group consisting of: 19G6D6, 36G7B8, 13F1A7,9F12D9, and 17C5C2 or competes against the reference antibody frombinding to the epitope.

In some embodiments, the antibody comprises a heavy chain complementarydetermining region 1 (CDR1), a heavy chain complementary determiningregion 2 (CDR2), and a heavy chain complementary determining region 3(CDR3), which collectively are at least 85% identical to the respectiveheavy chain CDRs of the reference antibody; and/or wherein the antibodycomprises a light chain CDR1, a light chain CDR2, and a light chainCDR3, which collectively are at least 85% identical to the respectivelight chain CDRs of the reference antibody.

In another embodiment, the heavy chain CDR1, heavy chain CDR2, and heavychain CDR3 collectively comprise 10 or fewer amino acid mutationsrelative to the respective heavy chain CDRs of the reference antibody;and/or wherein the light chain CDR1, light chain CDR2, and light chainCDR3 collectively comprise 10 or fewer amino acid mutations relative tothe respective light chain CDRs of the reference antibody.

In one embodiment, the antibody comprises a heavy chain variable regionthat is at least 85% identical to the heavy chain variable region of thereference antibody and/or a light chain variable region that is at least85% identical to the light chain variable region of the referenceantibody.

In some embodiments, the heavy chain variable region comprises 10 orfewer amino acid residue mutations relative to the heavy chain variableregion of the reference antibody; and/or and a light chain variableregion that comprises 10 or fewer amino acid mutations relative to thelight chain variable region of the reference antibody.

In some examples, the anti-CD40 antibody disclosed herein may comprisethe same heavy chain variable region CDRs as the reference antibodyand/or the same light chain variable region CDRs as the referenceantibody.

Any of the anti-CD40 antibodies described herein may be a human antibodyor a humanized antibody. Also provided herein is a pharmaceuticalcomposition comprising any of the anti-CD40 antibodies described hereinand a pharmaceutically acceptable carrier.

Further, the instant disclosure features an isolated nucleic acid or setof nucleic acids which collectively encode any of the CD40 bindingmolecules or any of the anti-CD40 antibodies described herein. Thenucleic acid or set of nucleic acids may be located on one or twovectors, for example, expression vectors. Also provided herein are hostcells comprising such vector(s).

The instant disclosure also features pharmaceutical compositionscomprising one or more of the CD40-binding molecules described hereinand/or one or more of the anti-CD40 antibodies disclosed herein for usein treating a target disorder as described herein, or uses of suchCD40-binding molecules and/or anti-CD40 antibodies for manufacturing amedicament for use in treating the target disorder.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are charts showing binding activity of various anti-CD40antibody 383 IgG variants as indicated to different types of Fcγreceptors expressed on CHO-K1 cells at the various concentrations asindicated. The concentrations of each IgG variant, from left to right,are 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/ml. Thetwo rightmost bars (“2nd only” and “blank”) served as controls. FIG. 1A:binding affinity to FcγRI. FIG. 1B: binding affinity to FcγRII. FIG. 1C:binding affinity to FcγRIIB. FIG. 1D: binding affinity to FcγRIIIA

FIG. 2 is a chart showing stimulation of human CD40 activation in areporter assay by a number of anti-CD40 antibody 383 IgG variants asindicated by IL8 secretion. The concentrations of each IgG variant, fromleft to right, correspond to: 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, and 10 μg/ml.

FIGS. 3A-3D are charts showing binding activity of a number of anti-CD40antibody 383 IgG variants as indicated to different types of Fcγreceptors expressed on CHO-K1 cells. The concentrations of eachanti-CD40 antibody 383 IgG variant, from left to right, are 0.3 μg/ml, 1μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/mi. The two rightmost bars (“2ndonly” and “blank”) served as controls. FIG. 3A: binding affinity toFcγRIIB. FIG. 3B: binding affinity to FcγRI. FIG. 3C: binding affinityto FcγRIIA. FIG. 3D: binding affinity to FcγRIIIA

FIGS. 4A-4F are charts showing stimulation of human CD40 activationindicated by IL8 secretion in a reporter assay by a number of anti-CD40antibody 383 IgG variants in solution and/or in co-culture with FcγRIIBexpressing cells. The concentrations of each IgG variant, from left toright, correspond to: 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml,0.3 μg/ml, and 1 μg/mi. Dash line indicates baseline reporter signal ineach assay. FIG. 4A: IgG variants IgG1m2, IgG2m20, IgG1m40, IgG4m40.FIG. 4B: IgG variants IgG1m240, IgG2m2040, IgG4m41, IgG4m42, IgG2m43,IgG2m44, and IgG4mAA. FIG. 4C: IgG variants IgG1 N297A, IgG2mAA, IgG2G4,IgG4mPE, and IgG1mAAG. FIG. 4D: IgG variants of IgG1m47, IgG1m48,IgG1m49, IgG1m50, and IgG1mAA. FIG. 4E: IgG variants of IgG4m46 andIgG4m30. FIG. 4F: IgG variants of IgG1m27, IgG2m19, IgG2 ml, IgG4m2 andIgG4m20.

FIGS. 5A-5D include a set of bar graphs showing the activity ofexemplary anti-CD40 antibody 383 IgG variants as indicated in activationof human dendritic cells (DC) from a healthy donor by the antibodieseither in solution (FIGS. 5A and 5B) or in co-culture of CHO cellsexpressing human FcγRIIB (FIGS. 5C and 5D). DC activation is indicatedby the bar graphs signal of IL-8 in the culture supernatant.

FIGS. 6A-6B are charts showing binding activity of anti-CD40 antibody383 IgG variants as indicated to human CD40 expressed on CHO-K1 cells.The concentrations of each IgG variant, from left to right, are 0.01μg/ml, 0.1 μg/ml, 1 μg/ml, and 10 μg/ml. The three rightmost bars(“Herceptin”, “2nd only” and “blank”) served as controls. FIG. 6A: anumber of anti-CD40 antibody 383 IgG variants as indicated. FIG. 6B: anumber of anti-CD40 antibody 383 IgG variants as indicated.

FIGS. 7A-7B are charts showing tumor growth curves of various anti-CD40antibody 383 IgG variants as indicated in a mouse tumor model. FIG. 7A:a chart showing tumor volume changes at various time points afterantibody treatment in homozygous B-hCD40 C57BL6 mice. The average±SEM oftumor sizes are shown. FIG. 7B: is a chart showing serum alaninetransaminase (ALT, a liver enzyme released into serum upon liver damage)level after treatment of antibodies as shown in homozygous B-hCD40C57BL6 mice. The average±SEM of tumor sizes are shown.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are CD40-binding molecules (e.g., CD40 agonists or CD40antagonists) comprising a CD40 binding moiety (e.g., an extracellulardomain of a CD40 ligand or an anti-CD40 antibody fragment) and anengineered (variant) Fc regions, which may have altered binding affinityand/or specificity to one or more Fc receptors, for example, enhancedbinding affinity to FcγRIIB, enhanced binding specificity to FcγRIIB,and/or substantially reduced binding affinity to one or more FcγRreceptors (e.g., low or no binding activity to all FcγR receptors).

Such CD40-binding molecules are expected to exhibit unexpected, superiortherapeutic activity. For example, CD40-binding molecules having avariant Fc region that has enhanced binding affinity to FcγRIIB would beexpected to exhibit enhanced agonistic activity and CD40-bindingmolecules having a variant Fc region that has enhanced bindingspecificity to FcγRIIB would be expected to exhibit higher tumorselectivity relative to the wild-type counterpart. Further, CD40-bindingmolecules having low or no binding affinity to FcγR receptors would helpactivate CD40-positive immune cells in tumor microenvironment and/orblock CD40-positive immune cells in immune disorders.

Accordingly, described herein are approaches for designing CD40-bindingmolecules comprising a CD40-binding moiety and an engineered Fc region(an Fc variant) which may be of an IgG molecule (e.g., IgG1, IgG2, andIgG4 molecules such as human IgG1, human IgG2, and human IgG4molecules), and uses thereof for modulating immune responses. Such an Fcvariant may have enhanced binding affinity to FcγRIIB (CD32B) relativeto the wild-type counterpart and/or binding selectivity as relative toother Fc receptors such as FcγRIII (CD16). Alternatively, such an Fcvariant may have substantially reduced binding affinity to one or moreFcγR receptors (e.g., to all FcγR receptors) relative to the wild-typecounterpart. “Substantially reduced” means that the binding affinity ofa Fc variant to a FcγR receptor is at least 60% lower (e.g., 70% lower,80% lower, 90% lower, 95% lower, 98% lower, or 99% lower) than thebinding affinity of the wild-type counterpart to the same FcγR receptor.In some examples, the Fc variant may have low or no binding affinity toall FcγR receptors, i.e., binding affinity cannot be detected byconventional assays or binding affinity is substantially low such thatno significant bioactivity would be triggered.

I. CD40-Binding Molecules

Described herein are CD40-binding molecules that comprise a CD40-bindingmoiety linked to an engineered Fc region having altered binding affinityand/or specificity to one or more Fc receptors as described herein. SuchCD40-binding molecules may be CD40 agonists, which are capable oftriggering the signaling mediated by CD40/CD40L upon binding to cellsurface CD40. Alternatively, the CD40-binding molecules described hereinare CD40 antagonists, which are capable of inhibiting the signalingmediated by CD40/CD40L upon binding to cell surface CD40. TheCD40-binding molecules are useful in modulating (selectively modulating)immune responses when administered to a subject in need of thetreatment.

(i) CD40-Binding Moieties

A CD40-binding moiety as described herein may be any peptide orpolypeptide that binds CD40, for example human CD40. CD40 is an immunecell receptor well known in the art. For example, NCBI GenBank AccessionNos. P25942.1 and AAB08705.1 provide information for human and mouseCD40, respectively. Provided below is an amino acid sequence of anexemplary human CD40 polypeptide.

Human CD40: (SEQ ID NO: 141)MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGEGVKQTATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ 

CD40 polypeptides from other species are known in the art and can beobtained from publicly available gene databases, for example, GenBank,using either the human sequence or the mouse sequence as a query.

In some embodiments, the CD40-binding moieties can be a polypeptidecomprising an extracellular domain of a CD40 ligand (CD40L or CD154).CD40L is a membrane glycoprotein expressed on the surface of T cells.The molecule has been shown to stimulate B cell proliferation and thesecretion of immunoglobulins in the presence of cytokines. In addition,CD40L may induce cytokine production and tumoricidal activity inperipheral blood monocytes. The sequence of CD40L and its extracellulardomain are well known in the art. For example, NCBI GenBank AccessionNo. NP_000065.1 (extracellular domain, amino acids 47-261) providesinformation for human CD40L. Provided below is an amino acid sequence ofan extracellular domain of an exemplary human CD40L.

Human CD40L (extracellular domain): (SEQ ID NO: 140)HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQ IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLK SPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL 

CD40L polypeptides from other species are known in the art and can beobtained from publicly available gene databases, for example, GenBank,using the human sequence as a query.

In other embodiments, the CD40-binding moiety described herein cancomprise an anti-CD40 antibody. As used herein, the term “anti-CD40antibody” refers to any antibody capable of binding to a CD40polypeptide, which can be of a suitable source, for example, human or anon-human mammal (e.g., mouse, rat, rabbit, primate such as monkey,etc.).

The anti-CD40 antibodies described herein comprise a heavy chain thatcomprises a heavy chain variable domain, which is linked to any of theFc variants described herein, and optionally a light chain thatcomprises a light chain variable region and a light chain constantregion. The heavy chain variable region (V_(H)) and optionally the lightchain variable region (V_(L)) are usually involved in antigen (CD40 inthis case) binding.

The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, also known as “complementarity determining regions”(“CDR”), interspersed with regions that are more conserved, which areknown as “framework regions” (“FR”). Each V_(H) and V_(L) is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The extent of the framework region and CDRs can be preciselyidentified using methodology known in the art, for example, by the Kabatdefinition, the Chothia definition, the AbM definition, and/or thecontact definition, all of which are well known in the art. See, e.g.,Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877;Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al(1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit.17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs).

In some embodiments, the anti-CD40 antibody as described herein can bindand inhibit the activity of CD40 by at least 50% (e.g., 60%, 70%, 80%,90%, 95% or greater, including any increment therein). The apparentinhibition constant (Ki^(app) or K_(i,app)), which provides a measure ofinhibitor potency, is related to the concentration of inhibitor requiredto reduce enzyme activity and is not dependent on enzyme concentrations.The inhibitory activity of an anti-CD40 antibody described herein can bedetermined by routine methods known in the art.

The K_(i) ^(app) value of an antibody may be determined by measuring theinhibitory effect of different concentrations of the antibody on theextent of the reaction (e.g., enzyme activity); fitting the change inpseudo-first order rate constant (v) as a function of inhibitorconcentration to the modified Morrison equation (Equation 1) yields anestimate of the apparent Ki value. For a competitive inhibitor, theKi^(app) can be obtained from the y-intercept extracted from a linearregression analysis of a plot of K_(i) ^(app) versus substrateconcentration.

$\begin{matrix}{v = {A \cdot \frac{\begin{matrix}{\left( {\lbrack E\rbrack - \lbrack I\rbrack - K_{i}^{app}} \right) +} \\{\sqrt{\left( {\lbrack E\rbrack - \lbrack I\rbrack - K_{i}^{app}} \right)^{2} + {{4\lbrack E\rbrack} \cdot}}K_{i}^{app}}\end{matrix}}{2}}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

Where A is equivalent to v_(o)/E, the initial velocity (v_(o)) of theenzymatic reaction in the absence of inhibitor (I) divided by the totalenzyme concentration (E).

In some embodiments, the anti-CD40 antibody described herein may have aKi^(app) value of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50,40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM orless for the target antigen or antigen epitope. In some embodiments, anyof the anti-CD40 antibodies may be further affinity matured to reducethe Ki^(app) of the antibody to the target antigen or antigenic epitopethereof.

In some instances, the anti-CD40 antibody may suppress the signalingtriggered by CD40/CD40L interaction by at least 50% (e.g., 60%, 70%,80%, 90%, 95% or greater, including any increment therein). Suchinhibitory activity can be determined by conventional methods or theassays described herein.

The antibodies described herein can be murine, rat, human, or any otherorigin (including chimeric or humanized antibodies). Such antibodies arenon-naturally occurring, i.e., would not be produced in an animalwithout human act (e.g., immunizing such an animal with a desiredantigen or fragment thereof or isolated from antibody libraries).

In some embodiments, the anti-CD40 antibody is a humanized antibody,which may have one of more of the elements or characteristics describedbelow or elsewhere herein. Humanized antibodies refer to forms ofnon-human (e.g., murine) antibodies that are specific chimericimmunoglobulins, immunoglobulin chains, or antigen-binding fragmentsthereof that contain minimal sequence derived from non-humanimmunoglobulin. In general, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In some instances, thehumanized antibody may comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,or six) which are altered with respect to the original antibody, whichare also termed one or more CDRs “derived from” one or more CDRs fromthe original antibody. Humanized antibodies may also involve affinitymaturation.

Methods for constructing humanized antibodies are also well known in theart. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033(1989). In one example, variable regions of V_(H) and V_(L) of a parentnon-human antibody are subjected to three-dimensional molecular modelinganalysis following methods known in the art. Next, framework amino acidresidues predicted to be important for the formation of the correct CDRstructures are identified using the same molecular modeling analysis. Inparallel, human V_(H) and V_(L) chains having amino acid sequences thatare homologous to those of the parent non-human antibody are identifiedfrom any antibody gene database using the parent V_(H) and V_(L)sequences as search queries. Human V_(H) and V_(L) acceptor genes arethen selected.

The CDR regions within the selected human acceptor genes can be replacedwith the CDR regions from the parent non-human antibody or functionalvariants thereof. When necessary, residues within the framework regionsof the parent chain that are predicted to be important in interactingwith the CDR regions can be used to substitute for the correspondingresidues in the human acceptor genes.

In some embodiments, the anti-CD40 antibodies described hereinspecifically bind to the corresponding target antigen or an epitopethereof, e.g., CD40 antigen or epitope. An antibody that “specificallybinds” to an antigen or an epitope is a term well understood in the art.A molecule is said to exhibit “specific binding” if it reacts morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular target antigen than it does with alternativetargets. An antibody “specifically binds” to a target antigen or epitopeif it binds with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other substances. For example, anantibody that specifically (or preferentially) binds to an antigen(CD40) or an antigenic epitope therein is an antibody that binds thistarget antigen with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other antigens or other epitopes inthe same antigen. It is also understood with this definition that, forexample, an antibody that specifically binds to a first target antigenmay or may not specifically or preferentially bind to a second targetantigen. As such, “specific binding” or “preferential binding” does notnecessarily require (although it can include) exclusive binding. In someexamples, an antibody that “specifically binds” to a target antigen oran epitope thereof may not bind to other antigens or other epitopes inthe same antigen (i.e., only baseline binding activity can be detectedin a conventional method). In some embodiments, the anti-CD40 antibodiesdescribed herein specifically bind to CD40. Alternatively, or inaddition, the anti-CD40 antibody described herein specifically bindshuman CD40 or a fragment thereof as relative to the mouse counterpart,or vice versa (e.g., having a binding affinity at least 10-fold higherto one antigen than the other as determined in the same assay under thesame assay conditions). In other instances, the anti-CD40 antibodydescribed herein may cross-react to human and a non-human CD40 (e.g.,mouse), e.g., the difference in binding affinity to the human and thenon-human CD40 is less than 5-fold, e.g., less than 2-fold, orsubstantially similar.

In some embodiments, an anti-CD40 antibody as described herein has asuitable binding affinity for the target antigen (e.g., CD40) orantigenic epitopes thereof. As used herein, “binding affinity” refers tothe apparent association constant or K_(A). The K_(A) is the reciprocalof the dissociation constant (K_(D)). The anti-CD40 antibody describedherein may have a binding affinity (K_(D)) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹, 10⁻¹⁰ M, or lower for the target antigen or antigenicepitope. An increased binding affinity corresponds to a decreased K_(D).Higher affinity binding of an antibody for a first antigen relative to asecond antigen can be indicated by a higher K_(A) (or a smallernumerical value K_(D)) for binding the first antigen than the K_(A) (ornumerical value K_(D)) for binding the second antigen. In such cases,the antibody has specificity for the first antigen (e.g., a firstprotein in a first conformation or mimic thereof) relative to the secondantigen (e.g., the same first protein in a second conformation or mimicthereof; or a second protein). Differences in binding affinity (e.g.,for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5,10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold. Insome embodiments, any of the anti-CD40 antibodies may be furtheraffinity matured to increase the binding affinity of the antibody to thetarget antigen or antigenic epitope thereof.

Binding affinity (or binding specificity) can be determined by a varietyof methods including equilibrium dialysis, equilibrium binding, gelfiltration, ELISA, surface plasmon resonance, or spectroscopy (e.g.,using a fluorescence assay). Exemplary conditions for evaluating bindingaffinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005%(v/v) Surfactant P20). These techniques can be used to measure theconcentration of bound binding protein as a function of target proteinconcentration. The concentration of bound binding protein ([Bound]) isgenerally related to the concentration of free target protein ([Free])by the following equation:

[Bound]=[Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K_(A),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(A), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

Provided below is an example anti-CD40 antibody 383 (V_(H) and V_(L)amino acid sequences; CDRs are indicated in boldface):

V_(H):  (SEQ ID NO: 128)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTR DTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSS  V_(L): (SEQ ID NO: 129)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK Further exemplary anti-CD40 antibodies are provided below (CDRs are indicated in boldface).  19G6D6  V_(H):  (SEQ ID NO: 130)EVQLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNDISIYNQKFRGKATLTV DKSSSTAYMELRSLTSEDTAVYYCARRFAYWGQGTLVTVSA  V_(L):  (SEQ ID NO: 131)DVVMTQIPLSLPVSIGDQASISCRSSQSLVYSYGNTYLHWFLQKPGQSPKLLIYNVSNRFSGVPDRFSGSGS GTDFTLRISRVEAEDLGVYFCSQGTHVPWTEGGGTKLEIK 36G7B8  V_(H): (SEQ ID NO: 132)QVQLQQPGAELVRPGASVKLSCKASGYIFISYWIHWLKQRPGRGLEWIGRIDPNNGGTKYNEKFRYKASLTV DKSSSTAYMQLSSLTSEDSAVYYCTKGVITTLVAGDYWGQGTTLTVSS    V_(L): (SEQ ID NO: 133)DIQMTQSPASLSASLGETVSIECLASEDISNDLAWYQQKSGKSPQLLIYFVDRLLDGVPSRFSGSGSGTRHS LKISGMQPEDEADYFCQQSYKYPPTFGGGTKLELK  13F1A7  V_(H):  (SEQ ID NO: 134)EVQILETGGGLVKPGGSLRLSCATSGFNFNDSFMNWVRQAPGKGLEWVAQIRNKNYNYATYYTESLEGRVTI SRDDSKSRVYLQVSSLRAEDSAVYYCTSYYYDGFADYFDYWGQGVMVTVSS  V_(L): (SEQ ID NO: 135)EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGSSPKVLIYRTSNLASGVPVRFSGSGSGTSY SLTIGTMEAEDVATYYCQQGSSIPYTFGGGTKLEIK 9F12D9  V_(H):  (SEQ ID NO: 136)EVHLVESGGGLVQPGRSLKLSCAASGFTFTNYGLHWIRQAPTKGLEWVASISPSGGVTYYRDSVKGRFTISR DNGKTTLHLQMDSLRSEDTATYYCALPFLGWGGANWIAHWGQGTLVTVSS V_(L): (SEQ ID NO: 137)DIKVTQSPSFLSASVGDRATINCKASQNLNKYLNWYQQKPGEPPKLLIYNTDNLYTGIPSRFSGSGSVADFT LTISGLQPEDVATYFCMQHNVRRTFGGGTKLELK 17C5C2  V_(H):  (SEQ ID NO: 138)EVHILETGGGLVKPGGSLGLSCTTSGFNFNDYFMNWVRQAPGKGLEWVAQIRNGNYDYAAYYAESLEGRVTI SRDDSKSSVNLQVSSLRAEDTAIYYCTSYYYDGHFDYFDNWGHGVMVTVSS  V_(L): (SEQ ID NO: 139)DIKMTQSPSFLSASVGDSVTFTCKASQNIYIYLNWYQQKFGEAPKLLIYNTNNLQTGIPSRFSGSESGTVFT LTISSLQPEDVATYFCLQHSSRRTFGGGTKLELK 

In some embodiments, the anti-CD40 antibodies described herein bind tothe same epitope as any of the exemplary antibodies described herein orcompetes against the exemplary antibody from binding to the CD40antigen.

An “epitope” refers to the site on a target antigen that is recognizedand bound by an antibody. The site can be entirely composed of aminoacid components, entirely composed of chemical modifications of aminoacids of the protein (e.g., glycosyl moieties), or composed ofcombinations thereof. Overlapping epitopes include at least one commonamino acid residue. An epitope can be linear, which is typically 6-15amino acids in length. Alternatively, the epitope can be conformational.The epitope to which an antibody binds can be determined by routinetechnology, for example, the epitope mapping method (see, e.g.,descriptions below). An antibody that binds the same epitope as anexemplary antibody described herein may bind to exactly the same epitopeor a substantially overlapping epitope (e.g., containing less than 3non-overlapping amino acid residue, less than 2 non-overlapping aminoacid residues, or only 1 non-overlapping amino acid residue) as theexemplary antibody. Whether two antibodies compete against each otherfrom binding to the cognate antigen can be determined by a competitionassay, which is well known in the art.

In some embodiments, the anti-CD40 antibodies disclosed herein comprisethe same heavy chain and light chain CDRs as those in (a) SEQ ID NO: 128and SEQ ID NO: 129, (b) SEQ ID NO: 130 and SEQ ID NO: 131 (19G6D6); (c)SEQ ID NO: 132 and SEQ ID NO: 133 (36G7B8); (d) SEQ ID NO: 134 and SEQID NO: 135 (13F1A7); (e) SEQ ID NO: 136 and SEQ ID NO: 137 (9F12D9), or(f) SEQ ID NO: 138 and SEQ ID NO: 139 (17C5C2). In some examples, theanti-CD40 antibody comprises a heavy chain variable domain of SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, or SEQID NO:138, and/or a light chain variable domain of SEQ ID NO: 129, SEQID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ IDNO:139.

Also within the scope of the present disclosure are functional variantsof any one of the exemplary anti-CD40 antibodies as disclosed herein.Such functional variants are substantially similar to any one of theexemplary anti-CD40 antibodies, both structurally and functionally. Afunctional variant comprises substantially the same V_(H) and V_(L) CDRsas any one of the exemplary anti-CD40 antibodies. For example, it maycomprise only up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) aminoacid residue variations in the total CDR regions of the antibody(collectively) and binds the same epitope of CD40 with substantiallysimilar affinity (e.g., having a K_(D) value in the same order).Alternatively or in addition, the amino acid residue variations areconservative amino acid residue substitutions. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the anti-CD40 antibody described herein maycomprise heavy chain CDRs, which in combination (collectively) have atleast 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and anyincremental percent therein) sequence identity with the heavy chain CDRsin SEQ ID NO: 128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ IDNO:136, or SEQ ID NO:138. Alternatively or in addition, the anti-CD40antibody may comprises light chain CDRs, which collectively have atleast 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incrementalpercent therein) sequence identity with those in SEQ ID NO: 129, SEQ IDNO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:139.

In some embodiments, the anti-CD40 antibody described herein maycomprise a heavy chain variable domain that is at least 80% (e.g., 85%,90%, 95%, 96%, 97%, 98%, 99% and any incremental percent therein) to SEQID NOs: 128, 130, 132, 134, 136, or 138 and/or a light chain variabledomain that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% andany incremental percent therein) identical to SEQ ID NOs: 129, 131, 133,135, 137, or 139.

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of interest. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

(ii) Engineered Fc Regions

The CD40-binding molecules described herein comprise a modified heavychain constant region, in which the Fc domain is modified to modulateits binding affinity and/or specificity to Fc receptors such as FcγRs.

In some embodiments, the Fc variants in the CD40-binding moleculesdescribed herein have enhanced selectivity to FcγRIIB relative to itswild-type counterpart. An Fc fragment having selectivity to FcγRIIB,selectively binding to FcγRIIB, or specifically binding to FcγRIIB is aterm well understood in the art. A molecule is said to exhibit“selective binding” or “specific binding” if it reacts more frequently,more rapidly, with greater duration and/or with greater affinity with aparticular target antigen (e.g., an FcγRIIB receptor) than it does withalternative targets (e.g., FcγRIII receptors). An Fc fragment“specifically binds” to an Fc receptor if it binds with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other Fc receptors. For example, an Fc fragment thatspecifically (or preferentially) binds to FcγRIIB is an Fc fragment thatbinds this Fc receptor with greater affinity, avidity, more readily,and/or with greater duration than it binds to other Fc receptors. It isalso understood with this definition that, for example, an Fc fragmentthat selectively or specifically binds to a first Fc receptor may or maynot specifically or preferentially bind to a second Fc receptor. Assuch, “selective binding,” “specific binding” or “preferential binding”does not necessarily require (although it can include) exclusivebinding. In some examples, an Fc fragment that “selectively binds,” or“specifically binds” to a target Fc receptor (e.g., FcγRIIB) may notbind to other Fc receptors (i.e., binding not detectable by routinemethods). In other embodiments, the variant Fc fragment does not bind toany FcγRs.

Relative binding affinities of IgG1, IgG2, and IgG4 to different Fcreceptors are given in Table 1 below.

TABLE 1 Relative Binding Affinities of Human and Mouse Immunoglobulinsto Fc Receptors Human Mouse FcγR IgG1 IgG2 IgG4 FcγR IgG1 IgG2a IgG2bI + + + + − + + + + I − + + + + + + + + IIA (H131) + + + + + + +III + + + + + + IIA (R131) + + + + + + IIB + + +/− + +IIB + + + + + + + + IIIA + + + + + + IV − + + + + + (V158) IIIA(F158) + + +/− + +

The Fc variants described herein may have enhanced selectivity toFcγRIIB relative to their wild-type counterparts (the wild-type parentFc region in which mutations are introduced to produce the Fc variants).The relative binding activity to FcγRIIB versus another Fc receptor(e.g., FcγRIII) of such an Fc variant is higher than the relativebinding activity to FcγRIIB versus the other Fc receptor (e.g., FcγRIII)of the wild-type counterpart. The Fc variant may have enhanced bindingactivity to FcγRIIB and/or decreased binding activity to another Fcreceptor, for example, FcγRIII. In some embodiments, the Fc variantsdescribed herein may have decreased binding activity to both FcγRIIB andanother Fc receptor (for example, FcγRIII); however, the level ofdecreased binding activity to the other Fc receptor (e.g., FcγRIII) isgreater than the level of decreased binding activity to FcγRIIB.

In some embodiments, an Fc variant as described herein has a suitablebinding affinity for FcγRIIB, e.g., enhanced as compared with thewild-type parent Fc from which the Fc variant is derived. As usedherein, “binding affinity” refers to the apparent association constantor K_(A). The K_(A) is the reciprocal of the dissociation constant(K_(D)). The Fc variant described herein may have a binding affinity(K_(D)) of at least 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰ M, or lower forFcγRIIB. An increased binding affinity corresponds to a decreased K_(D).Higher affinity binding of an Fc fragment for a first Fc receptorrelative to a second Fc receptor can be indicated by a higher K_(A) (ora smaller numerical value K_(D)) for binding the first Fc receptor thanthe K_(A) (or numerical value K_(D)) for binding the second Fc receptor.In such cases, the Fc variant has specificity for the first Fc receptorrelative to the second Fc receptor. In some embodiments, the Fc variantsdescribed herein have a higher binding affinity (a higher K_(A) orsmaller K_(D)) to FcγRIIB as compared to the binding affinity to FcγRIII(either FcγRIIIA or FcγRIIIB) Differences in binding affinity (e.g., forspecificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10,15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold.

In some embodiments, the Fc variants described herein may be designed bymutating one or more amino acid residues in the wild-type of human IgG1,IgG2, or IgG4 Fc fragments in light of the amino acid residues in thecorresponding mouse IgG, for example, mouse IgG1. A sequence comparisonof human and mouse IgGs (hIgG and mIgG, respectively) is provided below(SEQ ID NOs: 60-64, from top to bottom, each representing a combinationof fragments 211-245, 260-278, and 320-332 of the corresponding Fcregion):

       21-       22-       23-       24-   ...26-        27-     ...32-       33-       12345678 9012345

0123456789012345...0123456 7 89012 3 45678...01234567 8 9012            Upper      

ower    CH2 hIgG1  VDKKVEPK-SCDKTHT

PAPELLGGPSVFLFPP...TCVVVDVSHEDPEVKFNWY...KCKVSNKALPAPIhIgG2  VDKTVERK-CC-V-E-

PAPPVA-GPSVFLFPP...TCVVVDVSHEDPEVQFNWY...KCKVSNKGLPAPIhIgG4  VDKRVESKYG----PP

PAPEFLGGPSVFLFPP...TCVVVDVSQEDPEVQFNWY...KCKVSNKGLPSSImIgG1  VDKKIVPR-DC--G

TVPEVS---SVFIFPP...TCVVVDISKDDPEVQFSWF...KCRVNSAAFPAPImIgG2a VDKKIEPRGPTIKP

PAPNLLGGPSVFIFPP...TCVVVDVSEDDPDVQISWF...KCKVNNKDLPAPI 

The amino acid sequences of wild-type murine IgG1 and IgG2 Fc fragments,as well as exemplary Fc variants with reduced FcγR binding are providedbelow:

Amino acid sequence of wild-type mouse IgG1 Fc fragment: (SEQ ID NO: 142)VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQ PREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSL TCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK  Amino acid sequence of wild-type mouse IgG2a Fc fragment: (SEQ ID NO: 143)VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMT KKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN HHTTKSFSRTPGK  Amino acid sequence of mutant mouse IgG1mDANA Fc fragment: (SEQ ID NO: 144)VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQ PREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSL TCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKS LSHSPGK   Amino acid sequence of mutant mouse IgG2a1mDANA Fc fragment: (SEQ ID NO: 145)VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEV HTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMT KKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHN HHTTKSFSRTPGK  

In some embodiments, the Fc variants described herein is a human IgG1,G2, or G4 Fc variants comprising one or more mutations (e.g., amino acidsubstitutions, deletions, or additions) in the hinge domain of an Fcfragment. Human IgGs contain a core motif of CPPC or CPSC in the hingedomain (positions 226-229 according to the EU index). Positions 216 to225 are deemed as the upper portion of the hinge domain and positions230-238 are deemed as the lower portion of the hinge domain. Thenumbering system used herein, unless explicitly indicated, is accordingto the EU index. In some examples, the one or more mutations can belocated in the upper portion of the hinge domain. Alternatively or inaddition, the one or more mutations can be located in the lower portionof the hinge domain. In some embodiments, the Fc region of any of theanti-CD40 molecules disclosed herein may comprise at least one mutationat any of positions 220-331, preferably at any of positions 228-329.Such Fc variants may have altered binding affinity and/or specificity toone or more of Fc receptors (e.g., FcγIIB, FcγRI, FcγRIIA, or FcγRIIIA)as compared with their native counterparts.

In some embodiments, the mutations to a human IgG Fc can be madeaccording to the corresponding amino acid residues in the hinge domainof mouse IgG1. For example, mouse IgG1 does not contain the GGP motif atpositions 236-238. Accordingly, one or more of the residues in this GGPmotif can be deleted from a human IgG1, IgG2, or IgG4 Fc fragment toproduce the Fc variants described herein.

Alternatively or in addition, the human Fc variants may contain one ormore amino acid substitutions in the upper portion, in the lowerportion, or both of the hinge domain. For example, the Fc variant maycomprise one or more amino acid substitutions at one or more ofpositions 233, 234, 235, and/or 236. Such an amino acid substitution maybe in combination with the deletion of one or more of the GGP motif(236-238) noted herein. These mutations may be introduced into a humanIgG2 or IgG4 Fc fragment to produce the Fc variants described herein. Insome examples, the Fc variants described herein contains a deletion atone or more of the positions 236-238 (e.g., 236, 237, 238 or anycombination thereof)

Any of the mutations in the hinge domain described herein may be incombination with a mutation (e.g., amino acid substitutions) at one ormore positions that are involved in interaction with an Fc receptor.Such positions include, but are not limited to, positions 265, 267, 273,297, and 327-331, or a combination thereof. Exemplary amino acidsubstitutions at those positions include D265A, S267E, V273E, N297A,L328F, P329G, A330S, and/or P331S.

In some embodiments, the Fc variants disclosed herein can be derivedfrom an IgG1 molecule (e.g., human IgG1) and contain one or moremutations at positions at one or more of positions 220, 226, 229,234-238, 265, 267, 297, and 327-331. For example, the Fc variant maycomprise a substitution or deletion within positions 234-238, asubstitution at position 265 (e.g., D265A), a substitution at position267 (e.g., S267E), a substitution at position 297 (e.g., N297A), asubstitution at position 328 (e.g., L328F), a substitution at position329 (e.g., P329G), or a combination thereof. Alternatively or inaddition, it may comprise a substitution at one or more of the positions220 (e.g., C220S), 226 (e.g., C226S), 229 (e.g., C229S), 327 (e.g.,A327G), 330 (e.g., A330S), and 331 (e.g., P331S). In some instances, theFc variants derived from an IgG1 molecule may comprise a deletion at oneor more of positions 236-238.

In some embodiments, the Fc variants disclosed herein can be derivedfrom an IgG2 molecule (e.g., human IgG2) and contain one or moremutations at one or more of positions 233-235, 237-238, 265-268, 273,297, 328, 330, and 331. In some examples, such a Fc variant may comprisecomprises a deletion within positions 237-238 (e.g., a deletion atposition 237 or a deletion of both positions 237 and 238), asubstitution at position 265 (e.g., D265A), a substitution at position267 (e.g., S267E), a substitution at position 297 (e.g., N297A), asubstitution at position 328 (e.g., L328F), or a combination thereof.Alternatively or in addition, the Fc variant derived from an IgG2molecule may comprise a substitution at one or more of positions233-235, 237, 238, 268, 273, 330, and 331 (e.g., P233E, V234A, V234L,A235L, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S).

In yet other embodiments, the Fc variants disclosed herein can bederived from an IgG4 molecule (e.g., human IgG4) and contain one or moremutations at one or more positions of 228, 233-238, 265, 267, 273, 297,and 328. In some examples, such a Fc variant may comprise a substitutionat position 228 (e.g., S228P), a substitution or deletion at any ofpositions 235-238 (e.g., a deletion at one of positions 236-238, forexample, position 236 and position 237), a substitution at position 265(e.g., D265A), a substitution at position 267 (e.g., S267E), asubstitution at position 273 (e.g., V273E), a substitution at position297 (e.g., N297A), a substitution at position 328 (e.g., L328F), or acombination thereof. Alternatively or in addition, the Fc variant maycomprise a substitution at one or more of positions 233-235 and 237(e.g., E233P, F234V, F234A, L235S, L235E, L235A, and G237A).

Fc variants derived from IgG2 or IgG4 molecules that contain one or moremutations at positions 265, 267, 273, 297, 328, and/or 329 are alsowithin the scope of the present disclosure. Such mutations may includeamino acid substitutions at one or more of these positions, for example,D265A, S267E, V273E, N297A, L328F, and/or P329G.

In some instances, any of the Fc variants disclosed herein may furthercomprise a mutation at position 309, for example, a substitution (e.g.,V309L). Such an Fc variant may be derived from an IgG2 molecule (e.g.,human IgG2). In some embodiments, the mutation at position 309 may be incombination with mutations at one or more of positions 234, 268, 330,and 331. Examples of such Fc variants include G2m43.

In some embodiments, an Fc variant described herein may comprise anamino acid sequence at least 85% identical (e.g., 90%, 95%, 98%, 99%, orabove) to that of its wild-type counterpart (e.g., the Fc fragment ofwild-type human IgG1, IgG2, or IgG4 described herein).

In one example, the amino acid residue substitutions in an Fc variantdescribed herein are conservative amino acid residue substitutions.

Provided below is a sequence alignment showing exemplary positions wheremutations can be introduced into hIgG1, hIgG2, and IgG4 to producevarious Fc variants for the present disclosure.

Sequence alignment of human IgG1 variants relative to wild-type human IgG1 (SEQ IDNOs:70-88, 163-168, and 215 from top to bottom):

Sequence alignment of human IgG2 variants relative to wild-type human IgG2 (SEQ IDNOs: 89-105 and 169-173, from top to bottom):

Sequence alignment of human IgG4 variants relative to wild-type human IgG4 (SEQ IDNOs: 65, 106-127, and 174-177, from top to bottom)

The amino acid sequences of wild-type human IgG1, IgG2, and IgG4 Fcfragments, and a number of exemplary hIgG1, hIgG2, and hIgG4 Fc variants(position 221 and onward based on EU numbering) are provided below:

Amino Acid Sequence of Wild-Type Human IgG1 Fc Fragment:

(SEQ ID NO: 1)VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 

Amino Acid Sequence of Wild-Type Human IgG2 Fc Fragment:

(SEQ ID NO: 2)VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 

Amino Acid Sequence of Wild-Type Human IgG4 Fc Fragment:

(SEQ ID NO: 3)VDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK 

Amino Acid Sequence of Human IgG4 S228P Fc Variant:

(SEQ ID NO: 4)VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK 

Amino Acid Sequences of Exemplary Human IgG1 Fc Variants:

G1m1:  (SEQ ID NO: 5)VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK  G1m2:  (SEQ ID NO: 6)VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1m-2:  (SEQ ID NO: 7)VDKKVEPKCCVECPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK   G1m-4:  (SEQ ID NO: 8)VDKKVEPKYGPPCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK   G1m5:  (SEQ ID NO: 9)VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1m7:  (SEQ ID NO: 10)VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1m8:  (SEQ ID NO: 11)VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1m9:  (SEQ ID NO: 12)VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK  G1m15:  (SEQ ID NO: 13)VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEKFNWYVDGVEVHNAK           TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK   G1m17:  (SEQ ID NO: 14)VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK  G1m18:  (SEQ ID NO: 15)VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK  G1m19:  (SEQ ID NO: 16)VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK   G1m25:  (SEQ ID NO: 17)VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1m27:  (SEQ ID NO: 18)VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1m28:  (SEQ ID NO: 19)VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1m29:  (SEQ ID NO: 20)VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1mAA: (SEQ ID NO: 21)VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1mAG: (SEQ ID NO: 22)VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1m40:  (SEQ ID NO: 160)VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1m45 (SEQ ID NO: 178)VDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1m240 (SEQ ID NO: 179)VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1 N297A (SEQ ID NO: 180)VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1m47  (SEQ ID NO: 181)VDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKgLPssIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G1m48  (SEQ ID NO: 182)VDKKVEPKSsDKTHTsPPsPAPELLGGsSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK   G1m49  (SEQ ID NO: 183)VDKKVEPKSCDKTHTsPPsPAPpvaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK  G1m50  (SEQ ID NO: 184)VDKKVEPKSCDKTHTCPPCPAPEfeGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK 

Amino Acid Sequences of Exemplary Human IgG2 Fc Variants:

G2m1:  (SEQ ID NO: 23)VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK  G2m-1:  (SEQ ID NO: 24)VDKTVERKSCDKTHTCPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK   G2m2:  (SEQ ID NO: 25)VDKTVERKCCVECPPCPAPPFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK G2m-4:  (SEQ ID NO: 26)VDKTVERKYGPPCPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK   G2m5:  (SEQ ID NO: 27)VDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEEQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK   G2m7:  (SEQ ID NO: 28)VDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK   G2m8:  (SEQ ID NO: 29)VDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK   G2m9:  (SEQ ID NO: 30)VDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK   G2m10:  (SEQ ID NO: 31)VDKTVERKCCVECPPCPAPEVSSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK   G2m15:  (SEQ ID NO: 32)VDKTVERKCCVECPPCPAPPVASVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEEQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK   G2m17:  (SEQ ID NO: 33)VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK   G2m18:  (SEQ ID NO: 34)VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK   G2m19:  (SEQ ID NO: 35)VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK   G2m20:  (SEQ ID NO: 36)VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK  G2m27:  (SEQ ID NO: 37)VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK   G2m28:  (SEQ ID NO: 38)VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK   G2m40:  (SEQ ID NO: 161)VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK   G2m43  (SEQ ID NO: 185)VDKTVERKCCVECPPCPAPPaaasSVFLFPPKPKDTLMISRTPEVTCVVVDVSaEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK  G2m44  (SEQ ID NO: 186)VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSqEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK  G2mAA (SEQ ID NO: 213)VDKTVERKCCVECPPCPAPPaaaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK  G2m2040  (SEQ ID NO: 187)VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK  G2G4  (SEQ ID NO: 188)VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK  

Amino Acid Sequences of Exemplary Human IgG4 Fc Variants:

G4m1:  (SEQ ID NO: 39)VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK   G4m-1:  (SEQ ID NO: 40)VDKRVESKSCDKTHTPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK  G4m2:  (SEQ ID NO: 41)VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK   G4m-2:  (SEQ ID NO: 42)VDKRVESKCCVEPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLGK   G4m3:  (SEQ ID NO: 43)VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK   G4m4:  (SEQ ID NO: 44)VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK   G4m5:  (SEQ ID NO: 45)VDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK  G4m7:  (SEQ ID NO: 46)VDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK  G4m8:  (SEQ ID NO: 47)VDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK   G4m9:  (SEQ ID NO: 48)VDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK   G4m10:  (SEQ ID NO: 49)VDKRVESKYGPPCPPCPAPEVSSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK  G4m17:  (SEQ ID NO: 50)VDKRVESKYGPPCPPCPAPEFLSVFLEPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK  G4m18:  (SEQ ID NO: 51)VDKRVESKYGPPCPPCPAPEFLSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK  G4m19:  (SEQ ID NO: 52)VDKRVESKYGPPCPPCPAPEFLSVFLEPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK  G4m20:  (SEQ ID NO: 53)VDKRVESKYGPPCPPCPAPEFLGSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK   G4m25:  (SEQ ID NO: 54)VDKRVESKYGPPCPPCPAPEFLGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK   G4m27:  (SEQ ID NO: 55)VDKRVESKYGPPCPPCPAPEFLGPSVFLEPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK   G4m28:  (SEQ ID NO: 56)VDKRVESKYGPPCPPCPAPEFLGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK  G4m29:  (SEQ ID NO: 57)VDKRVESKYGPPCPPCPAPEFLGPSVFLEPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK   G4m30:  (SEQ ID NO: 58)VDKRVESKYGPPCPPCPAPEFLPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK   G4mPE:  (SEQ ID NO: 59)VDKRVESKYGPPCPPCPAPEFEGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK  G4m40:  (SEQ ID NO: 162)VDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK  G4m41  (SEQ ID NO: 189)VDKRVESKYGPPCPPCPAPPVAGGPSVFLEPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK  G4m42  (SEQ ID NO: 190)VDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK  G4m46  (SEQ ID NO: 191)VDKRVESKYGPPCPPCPAPEaLGaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKaYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK  G4mAA  (SEQ ID NO: 192)VDKRVESKYGPPCPPCPAPEaaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK 

The Fc variants described herein may exhibit an enhanced bindingactivity to FcγRIIB as compared with the wild-type counterpart. Examplesinclude G2m2, G2m5, G2m7, G2m8, G2m9, G2m19, G2m44, G1m7, G1m9, G1m27,G1m45, G1m47, G1m49, G1m50, G4m7, G4m46 and G4mPE. Alternatively or inaddition, the Fc variants may have an enhanced selectivity to FcγRIIB ascompared with their wild-type counterparts, for example, G2m1, G2m20,G2m43, G2m44, G2G4, G2mAA, G1m2, G1m15, G1m17, G1m18, G1m19, G1m27,G1m28, G1m29, G4 m1, G4m2, G4m7, G4m8, G4m9, G4m20, G4m25, G4m27, G4m28,G4m30, G4m46 and G4mPE. Such Fc variants may retain the binding activityto FcRn. These Fc variants can be used for constructing therapeuticagents described herein capable of cross-linking immune receptors andFcγRIIB receptor.

Alternatively, certain Fc variants as described herein may haveselectivity to FcγRIIB and/or apparent low or no binding activity to anyFcγR. Examples include G1m2, G1m25, G1m40, G1mAAG, G1m240, G2 m1, G2m20,G2m40, G2m2040, G4m5, G4m18, G4m19, G4m20, G4m30, G4m40, G4m41, andG4m42. Such Fc variants may retain the binding activity to FcRn.Therapeutic agents (e.g., antibodies) containing such Fc variants may becapable of cross-linking immune receptors and FcγRIIB receptor due toavidity effect.

The changes of binding affinity/specificity of the exemplary Fc variantsas compared with their wild-type counterparts are provided in Tables 2-4below. “N/A” indicates no data available. When the binding activity ofan Fc variant is found to be “no change” as compared with the wild-typecounterpart, it means that there is no significant variation of thebinding activity between the Fc variant and the wild-type counterpart asindicated by the same assay under the same experimental conditions. Whenthe binding activity of an Fc variant is “increased” or “decreased” asrelative to its wild-type counterpart means that the binding activity ofthe Fc variant is higher or lower than that of the wild-type counterpartas determined by the same assay under the same experimental conditionsand the variation is significant (e.g., biologically significant) asknown to those skilled in the art. When the binding activity of an Fcvariant is “slightly increased” or “slightly decreased” as relative toits wild-type counterpart means that the binding activity of the Fcvariant is higher or lower than that of the wild-type counterpart asdetermined by the same assay under the same experimental conditions andthe variation is statistically significant but to a limited level (e.g.,up to 10%).

TABLE 2 FcγR Binding Activity of Human IgG1 Mutants as Relative toWild-Type Human IgG1 IgG1 Changes of Binding Activity Relative toWild-Type Counterparts Mutant FcγRI FcγRIIA(H131) FcγRIIA(R131) FcγRIIBFcγRIIC FcγRIII G1m1 Decreased Decreased Decreased Decreased DecreasedDecreased G1m2 Decreased Decreased Decreased Decreased DecreasedDecreased G1m-2 No change N/A N/A No change N/A No change G1m-4 Nochange N/A N/A No change N/A No change G1m5 Increased IncreasedIncreased No change Increased Slightly decreased G1m7 IncreasedIncreased Increased Increased Increased Slightly decreased G1m8Increased Slightly No change Increased Slightly Slightly decreasedincreased decreased G1m9 Increased Decreased Increased IncreasedIncreased No change G1m15 Decreased Decreased Decreased DecreasedDecreased Decreased G1m17 Decreased Decreased Decreased DecreasedDecreased Decreased G1m18 Decreased Decreased No change DecreasedDecreased Decreased G1m19 Decreased Increased Increased IncreasedIncreased Decreased G1m25 Decreased Decreased Decreased DecreasedDecreased Decreased G1m27 Decreased Decreased Decreased IncreasedDecreased Decreased G1m28 Decreased Decreased Decreased DecreasedDecreased Decreased G1m29 Decreased Decreased Increased IncreasedIncreased Decreased G1mAA Decreased N/A Decreased Decreased N/ADecreased G1mAAG Decreased N/A Decreased Decreased N/A Decreased G1m240Decreased N/A Decreased Decreased N/A Decreased G1m45 Decreased N/ADecreased Decreased N/A Decreased G1N297A Decreased N/A DecreasedDecreased N/A Decreased G1m47 Decreased N/A Decreased Decreased N/ADecreased G1m48 Decreased N/A Decreased Decreased N/A Decreased G1m49Decreased N/A Decreased Decreased N/A Decreased G1m50 Decreased N/ADecreased Decreased N/A Decreased

TABLE 3 FcγR Binding Activity of Human IgG2 Mutants as Relative toWild-Type Human IgG2 IgG2 Change of Binding Activity Relative toWild-Type Counterpart Mutant FcγRI FcγRIIA(H131) FcγRIIA(R131) FcγRIIBFcγRIIC FcγRIII G2m1 No change Decreased Decreased Decreased No changeNo change G2m2 Increased Decreased No change Increased No change Nochange G2m10 No change Decreased No change Slightly No change No changedecreased G2m5 Slightly Decreased No change Decreased No change Nochange increased G2m7 No change Slightly Increased Increased Slightly Nochange increased increased G2m8 No change Decreased No change No changeNo change No change G2m9 No change No change Increased IncreasedIncreased No change G2m-1 Increased N/A N/A Decreased N/A No changeG2m-4 Increased N/A N/A Decreased N/A No change G2m15 No change N/A N/ADecreased N/A No change G2m17 No change N/A Increased No changeIncreased No change G2m18 Slightly N/A No change Decreased No change Nochange increased G2m19 No change N/A Increased Increased No change Nochange G2m20 No change N/A No change Decreased No change No change G2m27Slightly N/A N/A Increased N/A No change increased G2m28 No change N/AN/A Increased N/A No change G2m2040 Decreased N/A Decreased DecreasedN/A Decreased G2m43 No change N/A Decreased Decreased N/A DecreasedG2m44 No change N/A Decreased No change N/A Decreased G2mAA No changeN/A Decreased No change N/A Decreased G2G4 Decreased N/A Decreased Nochange N/A Decreased

TABLE 4 FcγR Binding Activity of Human IgG4 Mutants as Relative toWild-Type Human IgG4 IgG4 Change of Binding Activity Relative toWild-Type Counterpart Mutant FcγRI FcγRIIA(H131) FcγRIIA(R131) FcγRIIBFcγRIIC FcγRIII G4m1 Decreased Increased No change Slightly Slightly Nochange decreased increased G4m2 Decreased No change Decreased SlightlyNo change No change decreased G4m10 Decreased No change DecreasedDecreased No change No change G4m20 Decreased No change DecreasedDecreased No change No change G4m3 Decreased No change No changeDecreased No change No change G4m7 No change Increased IncreasedIncreased Increased Slightly increased G4m8 No change No changeIncreased Slightly No change No change increased G4m9 No change Nochange Increased Increased Increased No change G4m17 Decreased No changeIncreased Increased Increased Increased G4m18 Decreased No change Nochange Decreased No change Increased G4m19 Decreased No change No changeDecreased No change Slightly increased G4m25 Decreased No changeIncreased Slightly No change Increased decreased G4m27 Decreased Nochange No change Decreased No change Slightly increased G4m28 DecreasedNo change Slightly Increased Increased No change increased G4m29Decreased No change Increased Increased Increased Slightly increasedG4m4 Decreased No change No change Decreased No change No change G4m-1No change N/A N/A No change N/A No change G4m-2 No change N/A N/ADecreased N/A No change G4mPE Decreased N/A N/A Slightly No change Nochange decreased G4m30 Decreased N/A No change Decreased No change Nochange G4m40 Decreased N/A Decreased Decreased N/A Decreased G4m41Decreased N/A Decreased Decreased N/A Decreased G4m42 Decreased N/ADecreased Decreased N/A Decreased G4mAA Decreased N/A DecreasedDecreased N/A Decreased G4m46 Decreased N/A Decreased Decreased N/ADecreased

An Fc variant as described herein can be designed following the guidanceprovided herein and produced via routine recombinant technology. Itsbinding affinity and specificity to various Fc receptors can bedetermined via routine methods. See also Examples below.

In specific examples, the CD40-binding molecules (anti-CD40 molecules)disclosed herein may maintain the hinge region of the IgG1 parent,contain one or more mutations (e.g., one or more deletions at positions236-238 in light of mouse IgG1 as discussed above) that lead to reducedbinding affinity to Fc receptors such as FcγR2B but maintain residualbinding activity to the Fc receptor. Exemplary Fc variants having suchproperties include, but are not limited to, G1m2, G4m20, G4m30 andG4m46. The CD40 binding moiety in such anti-CD molecules may be derivedfrom an agonistic anti-CD40 antibody. Such a CD40 binding moiety may bederived from the anti-CD40 antibody 383 disclosed herein. In someinstances, the CD40 binding moiety comprises the same V_(H) and V_(L)CDRs as antibody 383. In some examples, the CD40 binding moiety maycomprise the same V_(H) and/or V_(L) chains as antibody 383 (SEQ ID NOs:128 and 129, respectively). Alternatively, the CD40 binding moiety canbe a functional variant of antibody 383 as disclosed herein. Such CD40binding molecules would have a number of advantageous features asdemonstrated in the Examples below. Examples include enhanced anti-tumorefficacy with improved therapeutic window and reduced side effects(e.g., less liver toxicity) in vivo, which may be attributable to thebaseline CD40 agonistic activity and the residual FcγRIIB bindingactivity. Too high CD40 agonist activity and/or FcγRIIB binding activityare expected to induce side effects such as cell toxicity.

In other specific examples, the CD40-binding molecule disclosed hereinmay comprise a Fc variant domain derived from IgG2 (e.g., maintain theIgG2 hinge) and contain one or more mutations (e.g., deletions orsubstitutions) at one or more of positions 236-238 in light of the mousecounterpart to reduce binding affinity to Fc receptors, particularlybinding affinity to FcγRIIB. In some instances, such Fc variants havedeletions at one or more of positions 236-238. Alternatively or inaddition, the Fc variants may have substitutions at positions 234, 237,238, 268 or a combination thereof. Such Fc variants may contain furthermutations at one or more positions involved in binding to Fc receptors,for example, position 265 (e.g., D265A). The Fc variants may maintainresidual binding activity to a Fc receptor such as FcγRIIB (e.g., G2m20and G2m43). Alternatively, the Fc variants may not bind to Fc receptorssuch as FcγRIIB (e.g., G2m40). The CD40 binding moiety in such anti-CDmolecules may be derived from an agonistic anti-CD40 antibody. Such aCD40 binding moiety may be derived from the anti-CD40 antibody 383disclosed herein. In some instances, the CD40 binding moiety comprisesthe same V_(H) and V_(L) CDRs as antibody 383. In some examples, theCD40 binding moiety may comprise the same V_(H) and/or V_(L) chains asantibody 383 (SEQ ID NOs: 128 and 129, respectively). Alternatively, theCD40 binding moiety can be a functional variant of antibody 383 asdisclosed herein. Such CD40 binding molecules would have a number ofadvantageous features as demonstrated in the Examples below. Examplesinclude enhanced anti-tumor efficacy with improved therapeutic windowand reduced side effects (e.g., less liver toxicity) in vivo, which maybe attributable to the combination of baseline CD40 agonistic activityand the residual FcγRIIB binding activity (e.g., 383-IghuG2m20), or thecombination of relatively high CD40 agonistic activity and no binding toFcγRIIB (e.g., 383-IghuG2m40).

Besides the FACS binding assay described herein, the activity of Fcvariants for Fcγ receptors can be examined in alternative assays. Forexample, CD40 reporter assays with or without co-culture of FcγRIIBexpressing cells can be used to demonstrate effect of receptorcross-linking and activation. Because of avidity effect resulting fromsimultaneous binding of antibody to two targets (e.g., CD40 andFcγRIIB), this assay is more sensitive to detect FcγRIIB binding.Minimal or no apparent binding detectable by FACS may show positiveresults in the reporter assay.

II. Preparation of CD40-Binding Molecules Comprising Fc Variants

The CD40-binding molecules described herein may be prepared byconventional methodology, for example, recombinant technology. Someexamples follow.

For CD40-binding molecules comprising an extracellular domain of aCD40L, a coding sequence of the CD40L extracellular domain can be fusedin frame with a coding sequence of a suitable Fc variant. The codingsequence for the whole CD40-binding molecule can be cloned into asuitable expression vector, which can be introduced into a suitable hostcell for protein expression.

Antibodies binding to CD40 can be prepared by any method known in theart. See, for example, Harlow and Lane, (1998) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York. In some embodiments,antibodies specific to CD40 or an extracellular domain thereof can bemade by the conventional hybridoma technology. The full-length targetreceptor or a fragment thereof, optionally coupled to a carrier proteinsuch as KLH, can be used to immunize a host animal for generatingantibodies binding to that antigen. The route and schedule ofimmunization of the host animal are generally in keeping withestablished and conventional techniques for antibody stimulation andproduction, as further described herein. General techniques forproduction of mouse, humanized, and human antibodies are known in theart and are described herein. It is contemplated that any mammaliansubject including humans or antibody producing cells therefrom can bemanipulated to serve as the basis for production of mammalian, includinghuman hybridoma cell lines. Typically, the host animal is inoculatedintraperitoneally, intramuscularly, orally, subcutaneously,intraplantar, and/or intradermally with an amount of immunogen,including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the anti-immune cell receptor monoclonal antibodiesdescribed herein. The hybridomas are expanded and subcloned, if desired,and supernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies capable of modulating the activity of the targetimmune cell receptor. Hybridomas that produce such antibodies may begrown in vitro or in vivo using known procedures. The monoclonalantibodies may be isolated from the culture media or body fluids, byconventional immunoglobulin purification procedures such as ammoniumsulfate precipitation, gel electrophoresis, dialysis, chromatography,and ultrafiltration, if desired. Undesired activity if present, can beremoved, for example, by running the preparation over adsorbents made ofthe immunogen attached to a solid phase and eluting or releasing thedesired antibodies off the immunogen Immunization of a host animal witha target antigen or a fragment containing the target amino acid sequenceconjugated to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N═C═NR,where R and R1 are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g.,produced by a hybridoma) may be sequenced and the polynucleotidesequence may then be cloned into a vector for further construction ofthe anti-CD40 antibodies described herein. The sequence encoding theantibody of interest may be maintained in vector in a host cell and thehost cell can then be expanded and frozen for future use.

In an alternative, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity(affinity maturation), or other characteristics of the antibody. Forexample, the constant region may be engineered to more resemble humanconstant regions to avoid immune response if the antibody is used inclinical trials and treatments in humans. It may be desirable togenetically manipulate the antibody sequence to obtain greater affinityto the target antigen and greater efficacy in inhibiting or activatingthe activity of the immune cell receptor. It will be apparent to one ofskill in the art that one or more polynucleotide changes can be made tothe antibody and still maintain its binding specificity to the targetreceptor.

In other embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse® fromAmgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.). In another alternative, antibodies maybe made recombinantly by phage display or yeast technology. See, forexample, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150;and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455.

Alternatively, antibody library technology, such as the phage displaytechnology (McCafferty et al., (1990) Nature 348:552-553), yeast displaytechnology, or mammalian cell display technology, can be used toisolated antibodies such as human antibodies specific to a target immunereceptor.

Methods for constructing humanized antibodies are also well known in theart. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033(1989). In one example, variable regions of V_(H) and V_(L) of a parentnon-human antibody are subjected to three-dimensional molecular modelinganalysis following methods known in the art. Next, framework amino acidresidues predicted to be important for the formation of the correct CDRstructures are identified using the same molecular modeling analysis. Inparallel, human V_(H) and V_(L) chains having amino acid sequences thatare homologous to those of the parent non-human antibody are identifiedfrom any antibody gene database using the parent V_(H) and V_(L)sequences as search queries. Human V_(H) and V_(L) acceptor genes arethen selected.

The CDR regions within the selected human acceptor genes can be replacedwith the CDR regions from the parent non-human antibody or functionalvariants thereof. When necessary, residues within the framework regionsof the parent chain that are predicted to be important in interactingwith the CDR regions (see above description) can be used to substitutefor the corresponding residues in the human acceptor genes.

Once an antibody capable of binding to a target immune cell receptor isobtained, the coding sequence of its heavy chain can be fused in-framewith the coding sequence of a suitable Fc variant, which may selectivelybind FcgRIIB or which does not bind any FcγRs, for example, any of theFc variants described herein via routine recombinant technology. In someinstances, the antibody is first investigated for its agonistic effectto activate the immune cell receptor to which it binds. Such anagonistic antibody can be selected for making the CD40-binding moleculesdescribed herein to enhance the agonistic effects.

In other instances, the antibody is first investigated for itsantagonistic effect to inhibit the immune cell receptor to which itbinds. Such an antagonistic antibody can be selected for making theCD40-binding molecule described herein to down-regulate immuneresponses. Alternatively, an Fc variant having low or no bindingactivity to any Fc receptor can be selected for making CD40 antagonists.

The resultant antibody molecules or CD40-binding molecules describedherein can be produced via routine recombinant technology as exemplifiedbelow. Nucleic acids encoding the heavy and light chain of an antibodyor the polypeptide of a CD40-binding molecule as described herein can becloned into one expression vector, each nucleotide sequence being inoperable linkage to a suitable promoter. In one example, each of thenucleotide sequences encoding the heavy chain and light chain is inoperable linkage to a distinct prompter. Alternatively, the nucleotidesequences encoding the heavy chain and the light chain can be inoperable linkage with a single promoter, such that both heavy and lightchains are expressed from the same promoter. When necessary, an internalribosomal entry site (IRES) can be inserted between the heavy chain andlight chain encoding sequences.

In some examples, the nucleotide sequences encoding the two chains ofthe antibody are cloned into two vectors, which can be introduced intothe same or different cells. When the two chains are expressed indifferent cells, each of them can be isolated from the host cellsexpressing such and the isolated heavy chains and light chains can bemixed and incubated under suitable conditions allowing for the formationof the antibody.

Generally, a nucleic acid sequence encoding one or all chains of anantibody can be cloned into a suitable expression vector in operablelinkage with a suitable promoter using methods known in the art. Forexample, the nucleotide sequence and vector can be contacted, undersuitable conditions, with a restriction enzyme to create complementaryends on each molecule that can pair with each other and be joinedtogether with a ligase. Alternatively, synthetic nucleic acid linkerscan be ligated to the termini of a gene. These synthetic linkers containnucleic acid sequences that correspond to a particular restriction sitein the vector. The selection of expression vectors/promoter would dependon the type of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the antibodiesdescribed herein, including, but not limited to, cytomegalovirus (CMV)intermediate early promoter, a viral LTR such as the Rous sarcoma virusLTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E.coli lac UV5 promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promotersinclude those using the lac repressor from E. coli as a transcriptionmodulator to regulate transcription from lac operator-bearing mammaliancell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those usingthe tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc.Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human GeneTherapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad.Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16or p65 using astradiol, RU486, diphenol murislerone, or rapamycin.Inducible systems are available from Invitrogen, Clontech and Ariad.

Regulatable promoters that include a repressor with the operon can beused. In one embodiment, the lac repressor from E. coli can function asa transcriptional modulator to regulate transcription from lacoperator-bearing mammalian cell promoters [M. Brown et al., Cell,49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl.Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor(tetR) with the transcription activator (VP 16) to create atetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP16), with the tetO-bearing minimal promoter derived from the humancytomegalovirus (hCMV) major immediate-early promoter to create atetR-tet operator system to control gene expression in mammalian cells.In one embodiment, a tetracycline inducible switch is used. Thetetracycline repressor (tetR) alone, rather than the tetR-mammalian celltranscription factor fusion derivatives can function as potenttrans-modulator to regulate gene expression in mammalian cells when thetetracycline operator is properly positioned downstream for the TATAelement of the CMVIE promoter (Yao et al., Human Gene Therapy). Oneparticular advantage of this tetracycline inducible switch is that itdoes not require the use of a tetracycline repressor-mammalian cellstransactivator or repressor fusion protein, which in some instances canbe toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)), to achieve its regulatable effects.

Additionally, the vector can contain, for example, some or all of thefollowing: a selectable marker gene, such as the neomycin gene forselection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; SV40 polyoma origins ofreplication and ColE1 for proper episomal replication; internal ribosomebinding sites (IRESes), versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Suitable vectors and methods for producing vectors containing transgenesare well known and available in the art.

Examples of polyadenylation signals useful to practice the methodsdescribed herein include, but are not limited to, human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) comprising nucleic acidsencoding any of the antibodies may be introduced into suitable hostcells for producing the antibodies. The host cells can be cultured undersuitable conditions for expression of the antibody or any polypeptidechain thereof. Such antibodies or polypeptide chains thereof can berecovered by the cultured cells (e.g., from the cells or the culturesupernatant) via a conventional method, e.g., affinity purification. Ifnecessary, polypeptide chains of the antibody can be incubated undersuitable conditions for a suitable period of time allowing forproduction of the antibody.

In some embodiments, methods for preparing an antibody described hereininvolve a recombinant expression vector that encodes both the heavychain and the light chain of an antibody as described herein. Therecombinant expression vector can be introduced into a suitable hostcell (e.g., a dhfr− CHO cell) by a conventional method, e.g., calciumphosphate-mediated transfection. Positive transformant host cells can beselected and cultured under suitable conditions allowing for theexpression of the two polypeptide chains that form the antibody, whichcan be recovered from the cells or from the culture medium. Whennecessary, the two chains recovered from the host cells can be incubatedunder suitable conditions allowing for the formation of the antibody.

In one example, two recombinant expression vectors are provided, oneencoding the heavy chain of the anti-immune cell receptor antibody andthe other encoding the light chain of the same antibody. Both of the tworecombinant expression vectors can be introduced into a suitable hostcell (e.g., dhfr− CHO cell) by a conventional method, e.g., calciumphosphate-mediated transfection. Alternatively, each of the expressionvectors can be introduced into a suitable host cells. Positivetransformants can be selected and cultured under suitable conditionsallowing for the expression of the polypeptide chains of the antibody.When the two expression vectors are introduced into the same host cells,the antibody produced therein can be recovered from the host cells orfrom the culture medium. If necessary, the polypeptide chains can berecovered from the host cells or from the culture medium and thenincubated under suitable conditions allowing for formation of theantibody. When the two expression vectors are introduced into differenthost cells, each of them can be recovered from the corresponding hostcells or from the corresponding culture media. The two polypeptidechains can then be incubated under suitable conditions for formation ofthe antibody.

Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recovery of the antibodiesfrom the culture medium. For example, some antibodies can be isolated byaffinity chromatography with a Protein A or Protein G coupled matrix.

The bioactivity of the antibodies described herein can be verified usingassays known in the art or described herein.

III. Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions comprisingthe CD40-binding molecules or any of the anti-CD40 antibodies describedherein and uses of such for modulating immune responses triggered byCD40/CD40L signaling. Such CD40-binding molecules as described herein oranti-CD40 antibodies as also described herein can be used for treatingdiseases such as cancer or immune-related disorders.

The CD40-binding molecules or anti-CD40 antibodies as described hereincan be mixed with a pharmaceutically acceptable carrier (excipient) toform a pharmaceutical composition for use in treating a target disease.“Acceptable” means that the carrier must be compatible with the activeingredient of the composition (and preferably, capable of stabilizingthe active ingredient) and not deleterious to the subject to be treated.Pharmaceutically acceptable excipients (carriers) including buffers,which are well known in the art. See, e.g., Remington: The Science andPractice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins,Ed. K. E. Hoover.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the antibodies (or the encoding nucleicacids) which can be prepared by methods known in the art, such asdescribed in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985);Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomescan be generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The CD40-binding molecules, anti-CD40 antibodies, or the encodingnucleic acid(s), may also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are known in the art, see, e.g., Remington, The Scienceand Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(v nylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g., Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an antibodywith Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulized by use of gases. Nebulized solutions may be breatheddirectly from the nebulizing device or the nebulizing device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner

IV. Therapeutic Applications

Any of the CD40-binding molecules or anti-CD40 antibodies disclosedherein may be used to modulating (e.g., enhancing or inhibiting) immuneresponses against invading pathogens and/or diseased cells such ascancer cells.

To practice the method disclosed herein, an effective amount of thepharmaceutical composition described herein can be administered to asubject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, the antibodies as described herein can beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human Mammals include, but are not limited to,farm animals, sport animals, pets, primates, horses, dogs, cats, miceand rats.

In some instances, the subject is a human patient having or at risk fora cell-mediated disease or disorder, such as cancer including but notlimited to lung cancer, stomach cancer, liver cancer, breast cancer,skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladdercancer, or colorectal cancer. Further exemplary cancers include, but arenot limited to, breast cancer; biliary tract cancer; bladder cancer;brain cancer including glioblastomas and medulloblastomas; cervicalcancer; choriocarcinoma; colon cancer; endometrial cancer; esophagealcancer; gastric cancer; hematological neoplasms including acutelymphocytic and myelogenous leukemia, e.g., B Cell CLL; T-cell acutelymphoblastic leukemia/lymphoma; hairy cell leukemia; chronicmyelogenous leukemia, multiple myeloma; AIDS-associated leukemias andadult T-cell leukemia/lymphoma; intraepithelial neoplasms includingBowen's disease and Paget's disease; liver cancer; lung cancer;lymphomas including Hodgkin's disease and lymphocytic lymphomas;neuroblastomas; oral cancer including squamous cell carcinoma; ovariancancer including those arising from epithelial cells, stromal cells,germ cells and mesenchymal cells; pancreatic cancer; prostate cancer;rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer includingmelanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma,and squamous cell cancer; testicular cancer including germinal tumorssuch as seminoma, non-seminoma (teratomas, choriocarcinomas), stromaltumors, and germ cell tumors; thyroid cancer including thyroidadenocarcinoma and medullar carcinoma; and renal cancer includingadenocarcinoma and Wilms tumor. A subject having a cancer can beidentified by routine medical examination, e.g., laboratory tests, organfunctional tests, CT scans, or ultrasounds. In some embodiments, thesubject to be treated by the method described herein may be a humancancer patient who has undergone or is subjecting to an anti-cancertherapy, for example, chemotherapy, radiotherapy, immunotherapy, orsurgery.

In other instances, the subject is a human patient having or at risk foran immune-related disorder Immune-related disorders refer to adysfunction of the immune system, including autoimmune diseases,immunodeficiencies, and/or allergies. In one embodiment, theimmune-related disorder is an autoimmune disease. Examples ofimmune-related disorders include, but are not limited to, rheumatoidarthritis (RA), systemic lupus erythematosus (SLE), Myasthenia Gravis(MG), Graves' Disease, Idiopathic Thrombocytopenia Purpura (ITP),Guillain-Barre Syndrome, autoimmune myocarditis, MembraneGlomerulonephritis, diabetes mellitus, Type I or Type II diabetes,multiple sclerosis, Reynaud's syndrome, autoimmune thyroiditis,gastritis, Celiac Disease, Vitiligo, Hepatitis, primary biliarycirrhosis, inflammatory bowel disease, spondyloarthropathies,experimental autoimmune encephalomyelitis, immune neutropenia, juvenileonset diabetes, and immune responses associated with delayedhypersensitivity mediated by cytokines, T-lymphocytes typically found intuberculosis, sarcoidosis, and polymyositis, polyarteritis, cutaneousvasculitis, pemphigus, pemphigold, Goodpasture's syndrome, Kawasaki'sdisease, systemic sclerosis, anti-phospholipid syndrome, Sjogren'ssyndrome, graft-versus-host (GVH) disease, and immune thrombocytopenia.A subject having an immune-related disorder can be identified by routinemedical examination, e.g., with laboratory tests. In some embodiments,the subject to be treated by the method described herein may be a humansubject with an immune-related disorder who has undergone or issubjecting to an immune-related disorder treatment, for example,immunosuppressive mediation, hormone replacement therapy, bloodtransfusions, anti-inflammatory medication, and/or pain medication.

As used herein, “an effective amount” refers to the amount of eachactive agent required to confer therapeutic effect on the subject,either alone or in combination with one or more other active agents. Insome embodiments, the therapeutic effect is modulating (e.g.,activating) the target immune receptor, thereby triggering or enhancingimmune responses mediated by the receptor. Determination of whether anamount of the antibody achieved the therapeutic effect would be evidentto one of skill in the art. Effective amounts vary, as recognized bythose skilled in the art, depending on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size, gender and weight,the duration of the treatment, the nature of concurrent therapy (ifany), the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of the individual components or combinations thereofbe used, that is, the highest safe dose according to sound medicaljudgment.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, moleculesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of a target disease/disorder. Alternatively, sustainedcontinuous release formulations of an antibody may be appropriate.Various formulations and devices for achieving sustained release areknown in the art.

In one example, dosages for a CD40-binding molecule or an anti-CD40antibody as described herein may be determined empirically inindividuals who have been given one or more administration(s) of theantibody. Individuals are given incremental dosages of the agonist. Toassess efficacy of the agonist, an indicator of the disease/disorder canbe followed.

Generally, for administration of any of the therapeutic agents such asthe CD40-binding molecule or an anti-CD40 antibody described herein, aninitial candidate dosage can be about 2 mg/kg. For the purpose of thepresent disclosure, a typical daily dosage might range from about any of0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate a target disease or disorder, or a symptom thereof. Anexemplary dosing regimen comprises administering an initial dose ofabout 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg ofthe antibody, or followed by a maintenance dose of about 1 mg/kg everyother week. However, other dosage regimens may be useful, depending onthe pattern of pharmacokinetic decay that the practitioner wishes toachieve. For example, dosing from one-four times a week is contemplated.In some embodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg(such as about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg,about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays. The dosing regimen(including the therapeutic used) can vary over time.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. In someexamples, the dosage of the therapeutic agents such as antibodiesdescribed herein can be 10 mg/kg. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history, as well as the properties of theindividual agents (such as the half-life of the agent, and otherconsiderations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of aCD40-binding molecule or anti-CD40 antibody as described herein willdepend on the specific antibody, antibodies, and/or non-antibody peptide(or compositions thereof) employed, the type and severity of thedisease/disorder, whether the CD40-binding molecule or anti-CD40antibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theagonist, and the discretion of the attending physician. Typically theclinician will administer a CD40-binding molecule or anti-CD40 antibody,until a dosage is reached that achieves the desired result. In someembodiments, the desired result is a decrease in thrombosis. Methods ofdetermining whether a dosage resulted in the desired result would beevident to one of skill in the art. Administration of one or moreCD40-binding molecule or anti-CD40 antibody can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of a CD40-binding molecule or anti-CD40 antibody may beessentially continuous over a preselected period of time or may be in aseries of spaced dose, e.g., either before, during, or after developinga target disease or disorder.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a target disease or disorder, a symptom of thedisease/disorder, or a predisposition toward the disease/disorder, withthe purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disorder, the symptom of the disease,or the predisposition toward the disease or disorder.

Alleviating a target disease/disorder includes delaying the developmentor progression of the disease, or reducing disease severity. Alleviatingthe disease does not necessarily require curative results. As usedtherein, “delaying” the development of a target disease or disordermeans to defer, hinder, slow, retard, stabilize, and/or postponeprogression of the disease. This delay can be of varying lengths oftime, depending on the history of the disease and/or individuals beingtreated. A method that “delays” or alleviates the development of adisease, or delays the onset of the disease, is a method that reducesprobability of developing one or more symptoms of the disease in a giventime frame and/or reduces extent of the symptoms in a given time frame,when compared to not using the method. Such comparisons are typicallybased on clinical studies, using a number of subjects sufficient to givea statistically significant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a target disease or disorder includes initialonset and/or recurrence.

In some embodiments, the antibodies described herein are administered toa subject in need of the treatment at an amount sufficient to activatethe activity of the target receptor by at least 20% (e.g., 30%, 40%,50%, 60%, 70%, 80%, 90% or greater) in vivo.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods. In some examples, thepharmaceutical composition is administered intraocularly orintravitreally.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipient is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

The particular dosage regimen, i.e., dose, timing and repetition, usedin the method described herein will depend on the particular subject andthat subject's medical history.

In some embodiments, more than one antibody, or a combination of anantibody and another suitable therapeutic agent, may be administered toa subject in need of the treatment. The CD40-binding molecule oranti-CD40 antibody can also be used in conjunction with other agentsthat serve to enhance and/or complement the effectiveness of the agents.

Treatment efficacy for a target disease/disorder can be assessed bymethods well-known in the art.

The therapeutic agent described herein may be utilized in conjunctionwith other types of therapy for the target disease such as cancer.Additional anti-cancer therapy includes chemotherapy, surgery,radiation, gene therapy, and so forth. When a second therapeutic agentis used, such an agent can be administered simultaneously orsequentially (in any order) with the CD40-binding molecule or anti-CD40antibody described herein.

When co-administered with an additional therapeutic agent, suitabletherapeutically effective dosages for each agent may be lowered due tothe additive action or synergy.

The treatments of the disclosure can be combined with otherimmunomodulatory treatments such as, e.g., therapeutic vaccines(including but not limited to GVAX, DC-based vaccines, etc.), orcheckpoint inhibitors (including but not limited to agents that blockCTLA4, PD1, LAGS, TIM3, etc.). Alternatively, the treatment of thepresent disclosure can be combined with a chemotherapeutic agent, forexample, pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,gemcitabine and cytarabine), purine analogs, folate antagonists andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitoticagents including natural products such as vinca alkaloids (vinblastine,vincristine, and vinorelbine), microtubule disruptors such as taxane(paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilonesand navelbine, epidipodophyllotoxins (etoposide, teniposide), DNAdamaging agents (actinomycin, amsacrine, anthracyclines, bleomycin,busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abciximab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) andgrowth factor inhibitors (e.g., fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylpednisolone, prednisone, and prenisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

For examples of additional useful agents see also Physician's DeskReference, 59.sup.th edition, (2005), Thomson P D R, Montvale N.J.;Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy20.sup.th edition, (2000), Lippincott Williams and Wilkins, BaltimoreMd.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine,15.sup.th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. TheMerck Manual of Diagnosis and Therapy, (1992), Merck ResearchLaboratories, Rahway N.J.

V. Kits

The present disclosure also provides kits for use in enhancing thedesired immune responses using any of the CD40-binding molecule oranti-CD40 antibody described herein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of thetherapeutic agent to treat, delay the onset, or alleviate a targetdisease as those described herein. The kit may further comprise adescription of selecting an individual suitable for treatment based onidentifying whether that individual has the target disease. In stillother embodiments, the instructions comprise a description ofadministering a therapeutic agent such as an antibody to an individualat risk of the target disease.

The instructions relating to the use of the therapeutic agent generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or alleviating a target disease ordisorder such as cancer. Instructions may be provided for practicing anyof the methods described herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is the therapeutic agent as those described herein.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1. Anti-CD40 Antibodies with Engineered Fc Regions

The cDNA sequences encoding the anti-CD40 antibody variable domain withvarious heavy chain CH1 and Fc region or human kappa light chainconstant region were synthesized and cloned. CHO transient expressionwas carried out with plasmids containing the corresponding heavy andlight chain sequences. These antibodies were purified by protein Aaffinity chromatography. The amino acid sequences of the heavy chain(HC) and the light chain (LC) are provided below:

383-huIgG LC (SEQ ID NO: 146)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTEGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 383-huIgG1m2 HC (SEQ ID NO: 147)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m27 HC(SEQ ID NO: 148)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2m1 HC(SEQ ID NO: 149)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2m19 HC(SEQ ID NO: 150)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2m20 HC(SEQ ID NO: 150)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG4m2 HC(SEQ ID NO: 152)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLIVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4m20 HC(SEQ ID NO: 153)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4m30 HC(SEQ ID NO: 154)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLIVDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK 383-msIgG LC(SEQ ID NO: 155)DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQANIFPLTEGGGIKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSENRNEC 383-msIgG1 DANA HC (SEQ ID NO: 156)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 383-msIgG1 HC(SEQ ID NO: 157)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 383-msIgG2a DANA HC(SEQ ID NO: 158)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 383-msIgG2a HC(SEQ ID NO: 159)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMIKKQVTLTCMVTDFMPEDIYVEWINNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTIKSFSRITGK

Additional Fc mutants were made and the amino acid sequences of theheavy chain (HC) are provided below (the light chain (LC) sequence isprovided above):

383-huIgG1 HC (SEQ ID NO: 207)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2 HC(SEQ ID NO: 208)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG4SP HC(SEQ ID NO: 209)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG1mAA(SEQ ID NO: 210)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m40 HC(SEQ ID NO: 193)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVaVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m45 HC(SEQ ID NO: 194)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m240 HC(SEQ ID NO: 195)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1 N297A(SEQ ID NO: 196)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m47 HC(SEQ ID NO: 197)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKgLPssIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m48 HC(SEQ ID NO: 198)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSsDKTHTsPPsPAPELLGGsSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m49 HC(SEQ ID NO: 199)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTsPPsPAPpvaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1m50 HC(SEQ ID NO: 214)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEfeGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG1mAAG(SEQ ID NO: 211)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2m40 HC(SEQ ID NO: 200)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2m43 HC(SEQ ID NO: 201)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCVECPPCPAPPaaasSVFLFPPKPKDTLMISRTPEVTCVVVDVSaEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2m44 HC(SEQ ID NO: 202)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSqEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2mAA HC(SEQ ID NO: 203)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCVECPPCPAPPaaaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2m2040 HC(SEQ ID NO: 204)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 383-huIgG2G4 HC(SEQ ID NO: 205)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4m40 HC(SEQ ID NO: 206)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4m41 HC(SEQ ID NO: 66)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4m42 HC(SEQ ID NO: 67)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4m46 HC(SEQ ID NO: 68)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPPCPPCPAPEaLGaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKaYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4mPE HC(SEQ ID NO: 212)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 383-huIgG4mAA HC(SEQ ID NO: 69)QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPPCPPCPAPEaaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Example 2. Determination of Binding Activity of CD40 Antibody with FcMutants to Cellular FcγR

To determine the binding activity of CD40 activity in IgG mutants tocellular Fc receptors, CHO cells were genetically engineered to expresshuman FcγRs (FcγRI, FcγRIIA, FcγRIIB, and FcγRIII) using a lentivirusdelivery system as known in the art.

Using anti-CD40 antibody 383 disclosed herein as an exemplary anti-CD40moiety, a number of anti-CD40 IgG Fc mutants, including 383-msIgG1,383-msIgG1 DANA, 383-msIgG2a, 383-msIgG2a DANA, 383-hIgG2, controlhuIgG1, 383-huIgG1m27, 383-huIgG2m19, 383-huIgG1m2, 383-huIgG2 m1,383-huIgG2m20, 383-huIgG4m2, 383-huIgG4m20, 383-huIgG4m30,383-huIgG1mAA, and 383-huIgG4SP (amino acid sequences provided above),were designed and constructed following the disclosures herein. TheseIgG mutants contain mutations in the upper hinge domain, the lower hingedomain, or within the CH2 domain.

For FACS analysis of the IgG mutants' binding to different FcγRs, FcγRoverexpressing CHO cells were harvested using trypsin-EDTA and weresuspended in cold staining buffer (3% BSA in PBS). Test IgG mutants,which were diluted in staining buffer, were added into the cells. Themixture was incubated at 4° C. for 2 hours, and then washed twice withcold staining buffer and re-suspended in PE-labeled anti-human IgGfollowed by incubation at 4° C. for 2 hours. The mixture was washedtwice with staining buffer and re-suspended in 2% PFA in PBS for FACS.

As shown in FIGS. 1A-1D and 3A-3D, a number of anti-CD40 human IgG1,IgG2, and IgG4 mutants showed binding activity to FcγRs expressed on thecell surface, whereas others exhibited no apparent finding to the FcγRsexamined. Summary of the binding property changes is listed in Tables2-4 above.

Example 3. CD40 Antibodies Having Engineered Fc Regions ShowedDifferential CD40 Agonist Activity

To determine the agonist activity of the IgG mutants, a CD40 reporterassay was developed, which involves reporter cells over-expressing humanCD40 T cells. This GS-H2-huCD40 reporter cells were re-suspended inassay buffer and cell density and viability were determined with trypanblue. The cell suspension was diluted to 1×10⁴ cells/mL with assaybuffer (MEM containing 1% FBS). The cells were added at 100 uL/well,such that the final cell number was 1000 cells/well in the assay plate(Nunc, Cat #167425). Serial working dilutions of the test samples wereprepared in assay buffer at 2× final concentrations. Samples were addedat 100 uL/well test sample at 2× final concentrations to the assayplate. The assay plate was incubated in 37° C., 5% CO₂ incubator for18-20 hours. After the 18-20 hour incubation, 8 ul of the supernatantfrom each well of the assay plate was collected and added to HTRFdetection assay plate (Nunc). A Human Interleukin 8 (reporter of CD40activation) detection assay was performed using a Human IL-8 Assay Kit(Cisbio, Cat #62IL8PEB). In particular, 16 ul assay volume was used. Theresults were read using Time Resolved Fluorescence by Tecan F200pro andthe relative light unit data was recorded.

As shown in FIG. 2 and FIGS. 4A-4E, all of the tested anti-CD40 antibodyIgG mutants stimulated human CD40 activation as evidenced by thesecretion of IL8. The magnitude of CD40 activation was influenced by theFc variants contained therein due to the hinge flexibility of eachantibody isotype structure.

CD40 reporter assay was also performed in coculture withFcγRIIB-expressing CHO cells and the results are shown in FIGS. 4A-4D.Binding to CD40 and FcγR2B by the tested antibody moleculessimultaneously in a microenvironment would affect individual binding dueto the avidity effect, which refers to the accumulated strength ofmultiple affinities of individual non-covalent binding interactions. Theantibodies showed increased activity correlating to their bindingactivity to FcγRIIB. It was noted that antibodies with Fc variants withweak or no apparent binding to FcγRIIB exhibited enhanced agonistactivity in co-culture reporter assay, suggesting higher sensitivity ofthe reporter assay which may be attributable to the avidity effect.Therefore, the Fc structure of the antibody can affect the agonistactivity of the antibodies with the same variable domains.

Example 4. Characterization of Anti-CD40 Antibody IgG Mutants inDendritic Cell (DC) Function Assays

The exemplary anti-CD40 antibody Ig variants comprising various Fcmutants were tested in vitro for CD40 binding activities and agonisticactivity as described in Examples above, as well as in vivo forantitumor efficacy and toxicity as in Example 6. Their activities inactivating human dendritic cells were carried out as following.

Frozen human PBMC from healthy donors (Allcells, Cat #PB005F) werethawed and transferred to a 50 ml tube with 25 mL RPMI1640 media.EasySep™ Human CD14 Positive Selection Kit II (Stemcell, Cat: 17858)were used to isolate B cells. Centrifuge the PBMC suspension for 15 minat 250 g, and discard the supernatant and resuspend the cells in 1.5 mLEasySep™ Buffer. Add Selection Cocktail to sample to a14 mL (17×100 mm)polystyrene round-bottom tube. 100 ul/ml, 150 ul for each sample.Transfer the cell from 50 ml tube to the 14 mL (17×100 mm) polystyreneround-bottom tube, mix and incubate for 15 min at RT. Mix the MagneticParticles before transfer to sample. Transfer Magnetic Particles tosample. 50 ul/ml, 75 ul for each sample. Mix and incubate for 10 mins atRT. Add 8.5 ml EasySep™ Buffer to top up the sample to 10 mL. Mix bygently pipetting up and down 2-3 times. Place the tube into the magnetand incubate for 5 min Pick up the magnet, and in one continuous motioninvert the magnet and tube, pouring off the supernatant. Collect thesupernatant for B cell Enrichment. Repeat the above wash steps foranother 2 times. Resuspend cells in 5 mL RPMI 1640. Count the celldensity.

The isolated CD14⁺ cells from human PBMCs were adjust to 1×10⁶ cell/mland add GM-CSF and IL-4 were added into the cell culture media. Incubatethe cells in a 37° C., 5% CO2 humidified incubator for 3 days. On day 3,the culture media were changed by transferring the culture supernatantto a centrifuge tube and then centrifuged at 250×g for 5 minutes. 10 mlfresh media containing GN-CSF and IL-4 were used to suspend the cellpellet in the centrifuge tube and then added to the flask during thecentrifugation so that the cell pellet thus formed would be resuspendedin the 10 ml fresh media. The resuspended cells were added back to thesame well or flask and incubated for an additional 2 days. On day 5, theabove steps were repeated for media change. The cells were furtherincubated for another additional 2 days. On day 7, immature dendriticcells can be observed and are ready to be used in the desiredapplication.

To perform DC Activation Assay, take medium with cells out of theT75-flask into a 50 ml tubes and centrifuge at 500×g for 5 min at RT.The supernatant was discarded and the cells were and re-suspend in 3 mLRPMI1640 10% FBS medium. Count the cell density and adjust the celldensity to 1e6/mL. 0.05 mL of the cell suspension were added to a 96well plate, 2.5×10⁴ cells/well. IL-8 and IL12 P70 were detected with theHuIL-12-P70 detection kit (Cisbio, Cat #: 62HIL12PEH) and HuIL-8detection kit (Cisbio, Cat #: 62HIL08PEH) in the cell culturesupernatant 24h later (day 3). The DC activation assay was alsoperformed in co-culture with FcγR2B expressing CHO cells.

As shown FIGS. 5A-5D, the tested CD40 antibody IgG mutants stimulatedhuman CD40 activation at various degrees as evidenced by the secretionof IL8 from the DC culture after antibody incubation. The magnitude ofDC activation was influenced by Fc variants contained therein due to thehinge flexibility of each antibody isotype structure and interactionwith Fc receptors expressed by the DC culture.

To ascertain the effect of Fc-FcγRIIB cross-linking for CD40 activation,DC activation was also performed in coculture with FcγR2B expressing CHOcells. Binding to CD40 and FcγRIIB by antibody molecules simultaneouslyin a microenvironment would affect individual binding due to avidityeffect leading to alteration of CD40 agonistic effect of the antibodies.The antibodies showed increased activity correlating to their bindingactivity to FcγRIIB, consistent to the observation in the reporter assaydescribed above. Therefore, the Fc structure of the antibody wouldaffect the overall agonist activity of the antibodies with the samevariable domains.

Example 5. CD40 Antibody in IgG Mutants Exhibit Similar Binding toCellular CD40

FACS were used to evaluate the binding properties of exemplary anti-CD40antibody IgG variants. CHO cells over-expressing human CD40 orcynomolgus monkey CD40 were harvested using trypsin-EDTA partialdigestion followed by centrifugation at 1000 rpm for 5 minutes. Thecells were resuspended in cold PBS-BSA (2%) at 5×10⁶/ml and aliquotedout to 100 ul/tube. The chimeric anti-CD40 antibodies were diluted inPBS-BSA in three times (final concentrations were 0.01, 0.1, 1, and 10ug/ml) and 50 ul of each concentration was added to the CHO-CD40 cells.The cell solutions were mixed and incubated at 4° C. in the dark for 2hours. The cells were then washed with PBS-BSA twice. Secondary antibodyconjugates (goat F(ab′)2 anti-human IgG-Fc (PE), pre-adsorbed (Abcam#ab98596)) at a concentration of 100 ul/vial was added and the cellswere mixed and incubated 4° C. in dark for 1 hour. The cells were thenwashed twice with PBS-BSA, followed by fixation in 2% PFA in PBS, andwere then subjected to FACS analysis with FACScaliber. As shown in FIGS.6A-6B, these antibodies exhibited high binding activity to human CD40expressed on the surface of CHO cells.

Example 6. CD40 Antibody in IgG Mutants Exhibit Differential Anti-TumorActivity in Animal Models in Vivo

Exemplary anti-CD40 antibodies with various Fc mutants were tested inmouse syngeneic tumor models in vivo to determine how Fc mutants couldaffect the efficacy and toxicity of these antibodies. C57BL6 mice withhuman CD40 extracellular domains knock in were used to develop syngeneicmouse tumor models. Murine colon cancer MC38 cells were subcutaneouslyimplanted into homozygous human CD40 knock-in C57BL6 mice on day 0. Micewere grouped when the tumor size was approximately 150±50 mm³ (n=6).CD40 antibodies were administered by intraperitoneal injections andtumor sizes were measure during 4-6 weeks of antibody treatment. Tumorsizes were measured by caliber 2 times a week and calculated as tumorvolume using formula of 0.5×length×width². Anti-tumor efficacy wasevaluated between tumor sizes of the control group and antibodytreatment group.

As shown in FIG. 7A, murine IgG2aDANA (Fc null) provided a referenceefficacy driven by Fab′2-induced CD40 activation. Human G4m2, G1m2,G2m20 and G2m40 showed efficacy superior or comparable to mIgG2aDANA.

The potential of antibody induced liver toxicity was examined in themouse model by measuring serum ALT level after antibody treatment. HumanG2 showed a very large ALT elevation with its value exceeded upper limitof the assay (>1000 U/L). The efficacious Fc mutants including G1m2,G2m20 and G2m40 showed less or minimal ALT elevation.

As examples, 383-G2m20 and -G2m40 showed higher agonist activity in theabsence of cross-linking and showed in vivo efficacy with FcγRinteraction (G2m40 is FcγR null in both binding and DC activationassays). Further, residual FcgRIIB binding activity of G2m20 enhancedefficacy. Fc variants G2 ml and G2m43 are expected to have similarefficacy but improved toxicity window given their similar in vitroactivity profiles to those of G2m20 and G2m40.

As another example, 383-G1m2 showed reduced agonist activity in theabsence of cross-linking. The residual FcgRIIB binding activity in vivopotentiated the in vivo agonist activity of this IgG variant butminimally on liver toxicity. Fc variants G1mAA, G1 N279A, G4m20, G4m30and G4m46 are expected to have similar efficacy and toxicity windowgiven their similar in vitro activity profiles to that of G1m2.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

1-68. (canceled)
 69. An anti-CD40 antibody, comprising (a) a heavy chainthat comprises a heavy chain variable (V_(H)) region, and (b) a lightchain that comprises a light chain variable (V_(L)) region; wherein theV_(H) region comprises the same heavy chain complementary determiningregions (CDRs) and the same light chain CDRs as antibody 383, whichcomprises a V_(H) of SEQ ID NO:128 and a V_(L) of SEQ ID NO:129; andwherein the heavy chain further comprises an engineered Fc region, whichcomprises at least one mutation at any of positions 228-329 as comparedto a wild-type Fc region counterpart, wherein the numbering is accordingto the EU index, and wherein the Fc region is of an IgG1 or IgG4. 70.The anti-CD40 antibody of claim 69, wherein the V_(H) region comprisesthe amino acid sequence of SEQ ID NO:128.
 71. The anti-CD40 antibody ofclaim 69, wherein the V_(L) region comprises the amino acid sequence ofSEQ ID NO:129.
 72. The anti-CD40 antibody of claim 69, wherein the V_(H)region comprises the amino acid sequence of SEQ ID NO:128 and the V_(L)region comprises the amino acid sequence of SEQ ID NO:129.
 73. Theanti-CD40 antibody of claim 69, wherein the antibody is a human antibodyor a humanized antibody.
 74. The anti-CD40 antibody of claim 69, whereinthe engineered Fc region is of an IgG1 and comprises: (a) an amino acidsubstitution or deletion within positions 233-238; (b) a substitution atone or more of positions 265, 267, 297, 328, and 329; or (c) acombination of (a) and (b).
 75. The anti-CD40 antibody of claim 74,wherein the engineered Fc region comprises a deletion at one or more ofpositions 236-238.
 76. The anti-CD40 antibody of claim 75, wherein theengineered Fc region further comprises a substitution at one or more ofpositions 233-235, 267, 273, 327, and
 328. 77. The anti-CD40 antibody ofclaim 74, wherein the engineered Fc region is G1m2, G1m15, G1m17, G1m18,G1m19, G1m25, G1m27, G1m28, G1m29, or G1m240.
 78. The anti-CD40 antibodyof claim 69, wherein the engineered Fc region is of an IgG4 andcomprises: (a) an amino acid substitution or deletion within positions235-238; (b) a substitution at one or more of positions 228, 265, 267,273, 297, and 328; or (c) a combination of (a) and (b).
 79. Theanti-CD40 antibody of claim 78, wherein the engineered Fc regioncomprises S228P substitution and a substitution or deletion at one ormore of positions 235-238.
 80. The anti-CD40 antibody of claim 78,wherein the engineered Fc region is selected from the group consistingof G4m2, G4m20, G4m28, G4m30, G4m41, G4m42, G4m46, G4mPE, G4mAA, andG4m40.
 81. A pharmaceutical composition, comprising the anti-CD40antibody of claim 69 and a pharmaceutically acceptable carrier.
 82. Amethod for modulating an immune response in a subject, the methodcomprising administering to a subject in need thereof the pharmaceuticalcomposition of claim
 81. 83. The method of claim 82, wherein the subjectis a human patient having or suspected of having cancer.
 84. The methodof claim 83, wherein the pharmaceutical composition is administered tothe subject by intravenous infusion.
 85. The method of claim 83, furthercomprising administering to the subject an effective amount of animmunomodulatory agent, a chemotherapeutic agent, or a combinationthereof.
 86. The method of claim 85, wherein the immunomodulatory agentis a therapeutic vaccine, or a checkpoint inhibitor.
 87. The method ofclaim 83, further comprising administering to the subject an effectiveamount of a checkpoint inhibitor, a chemotherapeutic agent, or acombination thereof.
 88. The method of claim 87, wherein the checkpointinhibitor is a PD1 inhibitor.